Multiplex amplification and separation of nucleic acid sequences on a bioelectronic microchip using asymmetric structures

ABSTRACT

A method for amplifying nucleic acids is provided wherein detection of amplified species is enhanced by the use of asymmetric amplification. Such amplification is made asymmetric by using divergent ratios of amplification primers or by using non-extending and/or non-cleavable amplification primers. Detection of the amplicons is improved because maintenance of single stranded species of amplicons during amplification facilitates their direct capture by immobilized probes without having to include denaturing steps.

FIELD OF THE INVENTION

[0001] This invention relates to devices, methods, and compositions ofmatter for performing active, multi-step, and multiplex nucleic acidsequence separation, amplification and diagnostic analyses. Generally,it relates to devices, methods, and compositions of matter foramplification and analysis of nucleic acid sequences in a sample. Morespecifically, the invention relates to methods, devices, andcompositions of matter for amplifying and analyzing nucleic acids usingnovel strand displacement amplification technologies in combination withbioelectronic microchip technology. The devices and methods of theinvention are useful in a variety of applications, including, forexample, disease diagnostics (infectious and otherwise), geneticanalyses, agricultural and environmental applications, drug discovery,pharmacogenomics, and food and/or water monitoring and analysis.

BACKGROUND OF THE INVENTION

[0002] The following description provides a summary of informationrelevant to the present invention. It is not an admission that any ofthe information provided herein is prior art to the presently claimedinvention, nor that any of the publications specifically or implicitlyreferenced are prior art to that invention.

DEFINITIONS

[0003] The following descriptions of the inventions contained herein usenumerous technical terms specific to the field of the invention.Generally, the meaning of these terms are known to those having skill inthe art and are further described as follows:

[0004] As used herein, “sample” refers to a substance that is beingassayed for the presence of one or more nucleic acids of interest. Thenucleic acid or nucleic acids of interest may be present in a mixture ofother nucleic acids. A sample, containing the nucleic acids of interest,may be obtained in numerous ways. It is envisioned that the followingcould represent samples: cell lysates, purified genomic DNA, body fluidssuch as from a human or animal, clinical samples, food samples, etc.

[0005] As used herein, the phrases “target nucleic acid” and “targetsequence” are used interchangeably. Both phrases refer to a nucleic acidsequence, the presence or absence of which is desired to be detected.Target nucleic acid can be single-stranded or double-stranded and, if itis double-stranded, it may be denatured to single-stranded form prior toits detection using methods, as described herein, or other well knownmethods. Additionally, the target nucleic acid may be nucleic acid inany form most notably DNA or RNA.

[0006] As used herein, “amplification” refers to the increase in thenumber of copies of a particular nucleic acid target of interest whereinsaid copies are also called “amplicons” or “amplification products”.

[0007] As used herein, “amplification components” refers to the reactionmaterials such as enzymes, buffers, and nucleic acids necessary toperform an amplification reaction to form amplicons or amplificationproducts of a target nucleic acid of interest.

[0008] As used herein, the phrase “multiplex amplification” refers tothe amplification of more than one nucleic acid of interest. Forexample, it can refer to the amplification of multiple sequences fromthe same sample or the amplification of one of several sequences in asample, as described in U.S. Pat. Nos. 5,422,252 and 5,470,723 which areincorporated herein by reference. The phrase also refers to theamplification of one or more sequences present in multiple sampleseither simultaneously or in step-wise fashion.

[0009] As used herein, “oligonucleotide” refers to a molecule comprisingtwo or more deoxyribonucleotides or ribonucleotides, preferably morethan three. The length of an oligonucleotide will depend on how it is tobe used. The oligonucleotide may be derived synthetically or by cloning.Oligonucleotides may also comprise protein nucleic acids (PNAs).

[0010] As used herein, “probe” refers to a known sequence of a nucleicacid that is capable of selectively binding to a target nucleic acid.More specifically, “probe” refers to an oligonucleotide designed to besufficiently complementary to a sequence of one strand of a nucleic acidthat is to be probed such that the probe and nucleic acid strand willhybridize under selected stringency conditions. Specific types ofoligonucleotide probes are used in various embodiments of the invention.For example, a “ligation probe” describes one type of probe designed tobind to both a target nucleic acid of interest and to an amplificationprobe. Additionally, a “ligated probe” or a “ligated probe template”refers to the end product of a ligation reaction between a pair ofligation probes.

[0011] As used herein, the terms “primer molecule” and “primer” are usedinterchangeably. A primer is a nucleic acid molecule with a 3′ terminusthat is either “blocked” and cannot be covalently linked to additionalnucleic acids or that is not blocked and possesses a chemical group atthe 3′ terminus that will allow extension of the nucleic acid chain suchas catalyzed by a DNA polymerase or reverse transcriptase.

[0012] As used herein, the phrase “amplification primer” refers to anoligonucleotide primer used for amplification of a target nucleic acidsequence.

[0013] The phrase “primer extension,” as used herein refers to the DNApolymerase induced extension of a nucleic acid chain from a freethree-prime (3′) hydroxy group thereby creating a strand of nucleic acidcomplementary to an opposing strand.

[0014] As used herein, the term “amplicon” refers to the product of anamplification reaction. An amplicon may contain amplified nucleic acidsif both primers utilized hybridize to a target sequence. An amplicon maynot contain amplified nucleic acids if one of the primers used does nothybridize to a target sequence. Thus, this term is used genericallyherin and does not imply the presence of amplified nucleic acids.

[0015] As used herein, “electronically addressable” refers to a capacityof a microchip to direct materials such as nucleic acids and enzymes andother amplification components from one position to another on themicrochip by electronic biasing of the capture sites of the chip.“Electronic biasing” is intended to mean that the electronic charge at acapture site or another position on the microchip may be manipulatedbetween a net positive and a net minus charge so that charged moleculesin solution and in contact with the microchip may be directed toward oraway from one position on the microchip or from one position to anotherposition.

[0016] As used herein, the phrase “capture site” refers to a specificposition on an electronically addressable microchip wherein electronicbiasing is initiated and where molecules such as nucleic acid probes andtarget molecules are attracted or addressed by such biasing.

[0017] As used herein, the term “anchored” refers to the immobilizationby binding of a molecule to a specified location on a microchip, such asa primer nucleic acid used in an SDA reaction, or a nucleic acid probeused to capture a target nucleic acid.

[0018] As used herein, the term “branched primer pair” refers to a pairof oligonucleotides that may be used as primers in an amplificationreaction and which are connected together through a chemical moiety suchthat the oligonucleotides are susceptible to hybridization and use asamplification primers.

[0019] As used herein, the term “primer capture probes” refers tooligonucleotides that are used to hybridize to selected target nucleicacids and provide anchoring support for such nucleic acids to a capturesite. Moreover, such oligonucleotides may function as amplificationprimers for amplifying said target nucleic acids.

[0020] As used herein, “hybridization” and “binding” are usedinterchangeably and refer to the non-covalent binding or “base pairing”of complementary nucleic acid sequences to one another. Whether or not aparticular probe remains base paired with a polynucleotide sequencedepends on the degree of complementarity, the length of the probe, andthe stringency of the binding conditions. The higher the stringency, thehigher must be the degree of complementarity, and/or the longer theprobe for binding or base pairing to remain stable.

[0021] As used herein, “stringency” refers to the combination ofconditions to which nucleic acids are subjected that cause doublestranded nucleic acid to dissociate into component single strands suchas pH extremes, high temperature, and salt concentration. The phrase“high stringency” refers to hybridization conditions that aresufficiently stringent or restrictive such that only specific basepairing will occur. The specificity should be sufficient to allow forthe detection of unique sequences using an oligonucleotide probe orclosely related sequence under standard Southern hybridization protocols(as described in J. Mol. Biol. 98:503 (1975)).

[0022] As used herein, “endonuclease” refers to enzymes (e.g.,restriction endonucleases, etc.) that cut DNA at sites within the DNAmolecule.

[0023] As used herein, a “restriction endonuclease recognition site”refers to a specific sequence of nucleotides in a double stranded DNAthat is recognized and acted upon enzymatically by a DNA restrictionendonuclease.

[0024] As used herein, the term “nicking” refers to the cutting of asingle strand of a double stranded nucleic acid by breaking the bondbetween two nucleotides such that the 5′ nucleotide has a free 3′hydroxyl group and the 3′ nucleotide has a 5′ phosphate group. It ispreferred that the nicking be accomplished with a restrictionendonuclease and that this restriction endonuclease catalyze the nickingof double stranded nucleic acid at the proper location within therestriction endonuclease recognition site.

[0025] As used herein, the phrase “modified nucleotide” refers tonucleotides or nucleotide triphosphates that differ in compositionand/or structure from natural nucleotide and nucleotide triphosphates.It is preferred that the modified nucleotide or nucleotide triphosphatesused herein are modified in such a way that, when the modifications arepresent on one strand of a double stranded nucleic acid where there is arestriction endonuclease recognition site, the modified nucleotide ornucleotide triphosphates protect the modified strand against cleavage byrestriction enzymes. Thus, the presence of the modified nucleotides ornucleotide triphosphates encourages the nicking rather than the cleavageof the double stranded nucleic acid.

[0026] As used herein, the phrase “DNA polymerase” refers to enzymesthat are capable of incorporating nucleotides onto the 3′ hydroxylterminus of a nucleic acid in a 5′ to 3′ direction thereby synthesizinga nucleic acid sequence. Examples of DNA polymerases that can be used inaccordance with the methods described herein include, E. coli DNApolymerase I, the large proteolytic fragment of E. coli DNA polymeraseI, commonly known as “Klenow” polymerase, “Taq” polymerase, T7polymerase, Bst DNA polymerase, T4 polymerase, T5 polymerase, reversetranscriptase, exo-BCA polymerase, etc.

[0027] As used herein, the term “displaced,” refers to the removing ofone molecule from close proximity with another molecule. In connectionwith double stranded oligonucleotides and/or nucleic acids, the termrefers to the rendering of the double stranded nucleic acid moleculesingle stranded, i.e., one strand is displaced from the other strand.Displacement of one strand of a double-stranded nucleic acid can occurwhen a restriction endonuclease nicks the double stranded nucleic acidcreating a free 3′ hydroxy which is used by DNA polymerase to catalyzethe synthesis of a new strand of nucleic acid. Alternatively, onenucleic acid may be displaced from another nucleic acid by the action ofelectronic biasing of an electrically addressable microchip.

[0028] As used herein, “ligase” refers to an enzyme that is capable ofcovalently linking the 3′ hydroxyl group of a nucleotide to the 5′phosphate group of a second nucleotide. Examples of ligases include E.coli DNA ligase, T4 DNA ligase, etc. As used herein, “ligating” refersto covalently attaching two nucleic acid molecules to form a singlenucleic acid molecule. This is typically performed by treatment with aligase, which catalyzes the formation of a phosphodiester bond betweenthe 5′ end of one sequence and the 3′ end of the other. However, in thecontext of the invention, the term “ligating” is also intended toencompass other methods of connecting such sequences, e.g., by chemicalmeans.

[0029] The term “attaching” as used herein generally refers toconnecting one entity to another. For example, oligomers and primers maybe attached to the surface of a capture site. With respect to attachingmechanisms, methods contemplated include such attachment means asligating, non-covalent bonding, binding of biotin moieties such asbiotinylated primers, amplicons, and probes to strepavidin, etc.

[0030] As used herein, the term “adjacent” is used in reference tonucleic acid molecules that are in close proximity to one another. Theterm also refers to a sufficient proximity between two nucleic acidmolecules to allow the 5′ end of one nucleic acid that is brought intojuxtaposition with the 3′ end of a second nucleic acid so that they maybe ligated by a ligase enzyme.

[0031] The term “allele specific” as used herein refers to detection,amplification or oligonucleotide hybridization of one allele of a genewithout substantial detection, amplification or oligonucleotidehybridization of other alleles of the same gene.

[0032] As used herein, the term “three-prime” or “3′” refers to aspecific orientation as related to a nucleic acid. Nucleic acids have adistinct chemical orientation such that their two ends are distinguishedas either five-prime (5′) or three-prime (3′). The 3′ end of a nucleicacid contains a free hydroxyl group attached to the 3′ carbon of theterminal pentose sugar. The 5′ end of a nucleic acid contains a freehydroxyl or phosphate group attached to the 5′ carbon of the terminalpentose sugar.

[0033] As used herein, the phrase “free three-prime (3′) hydroxylgroup,” refers to the presence of a hydroxyl group located at the 3′terminus of a strand of nucleic acid. The phrase also refers to the factthat the free hydroxyl is functional such that it is able to support newnucleic acid synthesis.

[0034] As used herein, the phrase “five-prime overhang” refers to adouble-stranded nucleic acid molecule, which does not have blunt ends,such that the ends of the two strands are not coextensive, and such thatthe 5′ end of one strand extends beyond the 3′ end of the opposingcomplementary strand. It is possible for a linear nucleic acid moleculeto have zero, one, or two, 5′ overhangs. The significance of a 5′overhang is that it provides a region where a 3′ hydroxyl group ispresent on one strand and a sequence of single stranded nucleic acid ispresent on the opposite strand. A DNA polymerase can synthesize anucleic acid strand complementary to the single stranded portion of thenucleic acid beginning from the free 3′ hydroxyl of the recessed strand.

[0035] As used herein, the term “bumper primer” refers to a primer usedto displace primer extension products in SDA reaction. The bumper primeranneals to a target sequence upstream of the amplification primer suchthat extension of the bumper primer displaces the downstreamamplification primer and its extension product.

[0036] As used herein, the terms “detected” and “detection” are usedinterchangeably and refer to the discernment of the presence or absenceof a target nucleic acid or amplified nucleic acid products thereof.

[0037] As used herein, “label” refers to a chemical moiety that providesthe ability to detect an amplification product. For example, a label ona nucleic acid may comprise a radioactive isotope such as ³²P ornon-radioactive molecule such as covalently or noncovalently attachedchromophores, fluorescent moieties, enzymes, antigens, groups withspecific reactivity, chemiluminescent moieties, and electrochemicallydetectable moieties.

[0038] The above definitions should not be understood to limit the scopeof the invention. Rather, they should be used to interpret the languageof the description and, where appropriate, the language of the claims.These terms may also be understood more fully in the context of thedescription of the invention. If a term is included in the descriptionor the claims that is not defined above, or that cannot be interpretedbased on its context, then it should be construed to have the samemeaning as it is understood by those of skill in the art.

BACKGROUND ART

[0039] Determining the nucleic acid sequence of genes is important inmany situations. For example, numerous diseases are caused by orassociated with a mutation in a gene sequence relative to the normalgene. Such mutation may involve the substitution of only one base foranother, called a “point mutation.” In some instances, point mutationscan cause severe clinical manifestations of disease by encoding a changein the amino acid sequence of the protein for which the gene codes. Forexample, sickle cell anemia results from such a point mutation.

[0040] Other diseases are associated with increases or decreases in copynumbers of genes. Thus, determining not only the presence or absence ofchanges in a sequence is important but also the quantity of suchsequences in a sample can be used in the diagnosis of disease or thedetermination of the risk of developing disease. Moreover, variations ingene sequences of both prokaryotic and eukaryotic organisms has proveninvaluable to identifying sources of genetic material (e.g., identifyingone human from another or the source of DNA by restriction fragmentlength polymorphism (RFLP)).

[0041] Certain infections caused by microorganisms or viruses may alsobe diagnosed by the detection of nucleic acid sequences peculiar to theinfectious organism. Detection of nucleic acid sequences derived fromviruses, parasites, and other microorganisms is also important where thesafety of various products is of concern, e.g., in the medical fieldwhere donated blood, blood products, and organs, as well as the safetyof food and water supplies are important to public health.

[0042] Thus, identification of specific nucleic acid sequences by theisolation of nucleic acids from a sample and detection of the sought forsequences, provides a mechanism whereby one can determine the presenceof a disease, organism or individual. Generally, such detection isaccomplished by using a synthesized nucleic acid “probe” sequence thatis complementary in part to the target nucleic acid sequence ofinterest.

[0043] Although it is desirable to detect the presence of nucleic acidsas described above, it is often the case that the sought for nucleicacid sequences are present in sample sources in extremely small numbers(e.g., less than 10⁷). The condition of small target molecule numberscauses a requirement that laboratory techniques be performed in order toamplify the numbers of the target sequences in order that they may bedetected. There are many well known methods of amplifying targetedsequences, such as the polymerase chain reaction (PCR), the ligase chainreaction (LCR), the strand displacement amplification (SDA), and thenucleic acid sequence-based amplification (NASBA), to name a few. Thesemethods are described generally in the following references: (PCR) U.S.Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; (LCR) EP Application No.,320,308 published Jun. 14, 1989; (SDA) U.S. Pat. Nos. 5,270,184, and5,455,166 and “Empirical Aspects of Strand Displacement Amplification”by G. T. Walker in PCR Methods and Applications, 3(1):1-6 (1993), ColdSpring Harbor Laboratory Press; and (NASBA) “Nucleic Acid Sequence-BasedAmplification (NASBA™)” by L. Malek et al. , Ch. 36 in Methods inMolecular Biology, Vol. 28: Protocols for Nucleic Acid Analysis byNonradioactive Probes, 1994 Ed. P. G. Isaac, Humana Press, Inc., Totowa,N.J. (Each of the above references are hereby incorporated byreference.)

[0044] With respect to analyzing and/or identifying target nucleic acidamplified products, i.e., “amplicons”, other well known techniques havebeen typically used including comparative size, relative migrationanalyses (e.g., Southern blot analysis) and hybridization analysis.However, comparative size or relative migration analyses are not optimalbecause they are undesirably slow and inaccurate. Additionally, whilehybridization analysis offers many advantages over these methods,hybridization analysis is neither rapid nor sensitive as compared to theteachings of the present invention.

[0045] With respect to PCR technology, since thermal cycling isrequired, PCR is not optimal for use in a microelectronic environmentbecause the heat fluctuations caused by the thermal cycling aredetrimental to the capture sites located on the surface of a microchip.Thermal cycling gives rise to other problems as well including therequirement for complex instrumentation (e.g., to ensure uniformheating, etc.), and, unacceptable time spans for completion of analysis(since each step must occur sequentially).

[0046] In contrast to PCR, the SDA technique is useful withmicroelectronic environments because it overcomes some of theabove-described undesirable characteristics of PCR, e.g., it is anisothermal. method and the amplification process is asynchronous, and,therefore, more rapid. Although the use of SDA has advantages over PCR,SDA schemes as currently practiced typically include the use ofsolution-based amplification protocols (e.g., disclosed in the abovementioned U.S. Pat. No. 5,455,166). Recent modifications of the SDAtechnique have advanced the technique to minimizing the number ofindividually designed primers for amplification as described in U.S.Pat. No. 5,624,825. However, such advances do not benefit fromenhancements realized in the current invention of electronicallycontrolled hybridization.

[0047] Other amplification procedures include the use of probes that arebound to a solid support. However, such procedures have not provided adiscemable advance in the art compared to the “anchored” SDA presentedherein and performed in conjunction with an electronically addressablemicrochip. For example, U.S. Pat. No. 5,380,489 discloses a method fornucleic acid amplification and detection of target nucleic acids whereinan adhesive element is used to affix capture probes so that targetmolecules may be more easily captured and detected. This method does notaddress the issue of simultaneous amplification, capture, and detectionas does the current invention. In another example, U.S. Pat. No.5,474,895 discloses detection of nucleic acids using a polystyrenesupport-based sandwich assay. Again, such a method merely involvespassive hybridization followed by subsequent detection followingsecondary passive hybridization of a probe.

[0048] Microchip arrays have also been used in association with nucleicacid amplification and detection. For example, miniaturized devices havebeen successfully developed for expression monitoring. See, e.g., M.Schena, et al., 270 Science 467-470 (1995), M. Schena, et al., 93 Proc.Natl Acad. Sci. USA 10614-619 (1996), J. DeRisi, et al., 14 Nat. Genet.457-60 (1996), R. A. Heller, et al., 94 Proc. Natl. Acad. Sci. USA2150-55 (1997), and J. DeRisi, et al., 278 Science 680-86 (1997).Miniaturized devices have also been successfully developed for analysisof single nucleotide polymorphisms (SNPs) within an amplicon. See, e.g.,Z. Guo, et al., 15 Nat. Biotechnol. 331-35 (1997), and E. Southern, 12Trends Genet. 110-15 (1996). (Each of the above publications are herebyincorporated by reference). These devices offer the potential forcombining the specificity of hybridization with the speed andsensitivity of microchip technology. However, none have successfullyprovided a suitable miniaturized device for the present purposes.

[0049] For example, although micro-devices have been used to analyzemultiple amplicons simultaneously (i.e., multiplex analysis), suchmultiplex analysis has been possible only if hybridization conditionsare compatible for each amplicon being tested. This detriment may bepartially compensated for by careful capture probe design, by the use ofvery long captures (e.g. cDNA for expression monitoring) (see, e.g., R.A. Heller, et al., (1997) supra, and M. Schena, et al., (1995) supra),or by extensive redundancy and overlap of shorter captureoligonucleotide sequences. However, taken together, these considerationshave imposed limitations on the use of most microchip devices. Moreover,high levels of redundancy such as that used with short oligonucleotidecapture sequences results in the requirement for large arrays andcomplex informatics programs to interpret data obtained, and stillcertain sequence-specific regions may remain difficult to analyze.Alternatively, the use of long capture oligonucleotides permits use ofuniformly elevated hybridization temperatures. However, the use of longcapture probes and elevated hybridization temperatures (e.g., in therange of 45 to 75° C.) largely precludes single base pair mismatchanalysis of highly related sequences.

[0050] Yet another disadvantage has become apparent with conventionalmicrochips (e.g., those disclosed in U.S. Pat. Nos. 5,202,231 and5,545,531, as well as in E. Southern et al., Genomics 13, 1008-1017(1992); M. Schena et al., Science 279, 467-470 (1995); M. Chee et al.,Science 274, 610-614 (1996); and D. J. Lockhart et al., NatureBiotechnology 14, 1675-1680 (1996) (all of which are herein incorporatedby reference)), in that they depend upon passive hybridization andsolution based amplification prior to the capture of amplified productson the microchips.

[0051] Further, many of these devices are unable to analyze and/ordetect the amplification of target molecules from multiple samplessimultaneously. In macroscopic devices, this latter problem isconventionally handled by “dot blot” formats in which individual samplesoccupy unique geometric positions with minimal contamination betweensamples. In contrast, for most microchips, the problem of detection andanalysis usually requires expensive and complex nucleic acid depositiontechnology similar to dot blot macroscopic deposition but on amicroscopic scale.

[0052] In another recent disclosure, (PCT WO96/01836), electronicmicrochips have been used in connection with PCR type amplification ofnucleic acids. However, an amplification system requiring thesimultaneous use of amplification enzymes and restriction enzymes forincreasing the quantity of target amplicons at a specific capture sitewas not contemplated nor possible in that system. Rather, restrictiondigestion of captured nucleic acid species was considered in connectionwith the removal of double stranded nucleic acid species from capturesites following PCR type amplification procedures with detection oftarget species occurring subsequent to enzymatic cleavage. Moreover,that system provided anchored amplification primers complementary toonly one strand of a target nucleic acid that were functional in a PCRreaction.

[0053] Like other microchip based amplification and detection platforms,the invention conceptualized in the PCT WO 96/01836 publication issubstantially limited as compared to the SDA on electronicallyaddressable microchips disclosed herein because the PCR typeamplification of target species as taught in that publication requiredrepeated disruption of double stranded species as well as functionalityof solution based reverse primers. Such a situation results in thereduction of efficient amplification due to primer-primer interactionswhile use of restriction enzymes is inhibited due to fluctuations inreaction buffer conditions.

[0054] Finally, other aspects of amplification and detection of nucleicacids have been problematic and/or not optimal. One such problem hasbeen the loss of specificity in the restriction endonuclease cleavage ofnucleic acids by restriction enzymes. For example, it is known that somerestriction endonucleases lose specificity for their DNA recognitionsequence with increased osmotic pressure or reduced water activity. C.R. Robinson et al. J. Mol. Biol. 234: 302-306 (1993). With reduced wateractivity, the restriction endonucleases will cleave DNA at recognitionsites that differ by one base pair from the normal recognition site. Therestriction sites that are off by one base pair are called “star” sitesand the endonucleases recognition and cleavage of these star sites iscalled “star activity.”

[0055] Robinson et al. found that bound water participates in sequencespecificity of EcoRI DNA cleavage (Biochemistry 33(13):3787-3793(1994)),and further found that increasing hydrostatic pressure by conducting thereactions at elevated pressure from 0 to 100 atm. inhibited andultimately eliminated star activity induced by osmotic pressure forEcoRI, PvuII, and BamHI, but not for EcoRV. (Proc. Natl. Acad. Sci. USA92:3444-3448 (1995)). One recurrent problem with SDA that relies onrestriction endonucleases is the frequency with which non-targetsequences are amplified in a primer-independent manner due to staractivity. Thus, there is a need to reduce or eliminate star activity inSDA reactions. In one embodiment of the current invention, we providefor the elimination of such star activity in SDA reactions byapplication of a high pressure SDA method.

[0056] In addition to advancing SDA technology by eliminating staractivity, we also provide for various other advancements in thedetection of nucleic acids using SDA in combination with a bioelectronicmicrochip. For example, amplification and separation of nucleic acidsequences may be carried out using ligation-dependent SDA. In contrastto ligation-dependent amplification procedures known in the art thatrequire the amplified products to be separated from the startingmaterial by a capture step, or that require that free ligation probe beseparated from bound probe prior to ligation, the current inventioneliminates the need to make separate isolation steps. Further, thecurrent invention improves upon the SDA amplification process byeliminating the need for bumper primers as they have been used in theart. For example, typical ligation-dependent amplification proceduresinclude capture steps by labeling one of the primers used duringamplification. Separation may occur prior to ligation to preventtemplate independent ligation of the primers or separation may occurfollowing ligation to isolate target DNA amplicons from thenon-labeled/amplified DNA. Target DNA amplicons containing this labelare separated from the non-labelled/amplified DNA. This separationrequires an extra step following amplification. This extra manipulationof the sample increases the complexity of the procedure and therebyrenders it less useful as a simple alternative to other current DNAamplification methods such as PCR. This extra manipulation of samplealso hinders automation of the amplification procedure. In oneembodiment of the current invention a ligation-dependent SDA method isprovided that eliminates such extra steps facilitating automation ofamplification and detection of target nucleic acids.

[0057] In other embodiments, we have provided additional advancements innucleic acid amplification and detection technology using SDA andelectronically addressable microchips which advancements collectivelyshow that a need remains for devices, methods, and compositions ofmatter for efficiently and optimally amplifying, detecting and analyzingtarget nucleic acid sequences of interest.

SUMMARY OF THE INVENTION

[0058] This invention relates broadly to devices, methods, andcompositions of matter for the multiplex amplification, detection andanalysis of nucleic acid sequences wherein the amplification, detectionand analysis is optimally accomplished using SDA in combination withbioelectronic microchip technology. The invention provides variousefficient and optimal methods of amplifying target nucleic acids ofinterest as well as methods for analyzing resultant amplicons. Inaddition, the invention enables the amplification and analysis (eithersequentially or simultaneously) of multiple samples containing targetnucleic acids on a single open bioelectronic microchip.

[0059] In one aspect of this invention, the microchip device is anelectronically controlled microelectrode array. See, PCT applicationWO96/01836, the disclosure of which is hereby incorporated by reference.In contrast to the passive hybridization environment of most othermicrochip devices, the electronic microchip devices (or activemicroarray devices) of the present invention offer the ability toactively transport or electronically address nucleic acids to discretelocations on the surface of the microelectrode array, and to bind theaddressed nucleic acid at those locations to either the surface of themicrochip at specified locations designated “capture sites” or tonucleic acids previously bound at those sites. See, R. Sosnowski, etal., 94 Proc. Natl. Acad. Sci. USA 119-123 (1997), and C. Edman, et al.,25 Nucleic Acids Res. 4907-14 (1997). The use of these activemicroarrays circumvent many of the limitations encountered by passivemicrodevices.

[0060] The active microchip arrays of the present invention overcome thesize dependency of capture oligonucleotides and the complexityrequirements of passive microdevices. Also, the microchip arrays of thepresent invention allow multiple independent sample analyses upon thesame open microarray surface by selectively and independently targetingdifferent samples containing nucleic acids of interest to variousmicroelectrode locations. In other words, they allow parallel multiplesample processing on an open array. As mentioned above, traditionalnucleic acid detection methodologies are restricted by the frequentlylong amplification and hybridization times required to achieveresolvable signals. An additional limitation to such methodologies isthe inability to carry out multiplex hybridization events upon theiranalytical surfaces, thereby restricting information obtainable in anyone assay. Both of these limitations are overcome in the presentinvention by use of active microelectronic arrays capable of selectivelytargeting and concentrating DNA to specific sites on the array. Afurther strength of these devices is the power to perform electronichybridization and denaturation to discriminate single basepolymorphisms. Thus, these active microelectrode arrays demonstrate theflexibility to handle a wide variety of tasks upon a common platform,beyond those seen with other microdevices.

[0061] The present invention preferably uses an amplification methoddifferent from traditional PCR. Specifically, the present invention usesstrand displacement amplification (SDA). SDA is an amplificationmethodology that has sufficient sensitivity and robustness to rapidly(e.g., in about 15-45 minutes) and exponentially amplify a small numberof target molecules over a complex background. See, e.g., C. Spargo, etal., 10 Molecular and Cellular Probes 247-56 (1996). In contrast to PCR,SDA is an isothermal technique that requires simpler thermal control andassociated instrumentation. SDA is more compatible with a unifiedamplification-hybridization-detection system (i.e. a system wherein allsteps are unified in one place, e.g., on a microarray chip) for rapidanalyses of nucleic acids. This is primarily due to the fact that SDAdoes not require conditions (e.g. thermal cycling) which could bedetrimental to the microarray of an electronically addressablemicrochip.

[0062] The efficiency of amplification reactions in passivehybridization wherein probes designed to capture target and ampliconnucleic acid molecules are anchored to the surface of the microarray islimited during the initial phases of amplification due to the lowfrequency of hybridization of target nucleic acid species to theappropriate primers located on the tethering surface. Typically, thisprocess requires hours, even in reduced volumes of solution. However,the efficiency of this process is dramatically increased byelectronically concentrating, (i.e. addressing), the nucleic acid to thevicinity of “anchored” primers, thereby increasing the frequency ofencounter between the solution phase target nucleic acid and theanchored primers. Whereas prior concepts used PCR in connection withonly one of the two amplification primers necessary for PCRamplification anchored to a specific site on the microarray, the currentinvention contemplates that both amplification primers necessary for SDAare anchored to a specific capture site on the microarray. Thus, in oneembodiment of the invention, electronically concentrating andhybridizing the target nucleic acid to the surface of a microchip (i.e.,capture sites) prior to the introduction of amplification reactionbuffers, enzymes, nucleotides, etc., benefits greatly “anchored”amplification reactions, such as “anchored SDA”, as described below. Therapid concentration and subsequent specific hybridization of templatenucleic acid molecules to their complementary anchored amplificationprimers permits the surface of the array to be washed, removing unwantedand possibly interfering non-target nucleic acid from the reactionenvironment.

[0063] Employing electronic addressing of target nucleic acids tospecific locations on the microarray has at least three other advantagesover prior passive hybridization technologies. First, the overall timeand efficiency of the amplification process is dramatically improvedsince a major rate-limiting step (that of the time required for thetemplate to find the anchored primers) is removed from the overallreaction rate. Also, the use of electronic addressing acts toelectronically concentrate target nucleic acids such that hybridizationof the target species to the anchored amplification probes increases thenumber of target molecules at the selected site as compared to thenumber of target molecules that would be found at any particular site ona non-electronic, passive hybridization microarray for an equivalenttime period. The result is that the absolute numbers of startingmolecules for the amplification process is dramatically increasedresulting in improvement in both the overall yield of amplificationproducts and the sensitivity to lower starting template numbers.

[0064] The second advantage is that discrete target nucleic acids can beapplied to specific locations upon the array surface thereby allowingmultiple, different nucleic acid samples to be simultaneously amplifiedon one array. Alternatively, a nucleic acid sample can be targeted toseveral different locations, each containing specific sets ofamplification primers so that multiple different amplification reactionscan be simultaneously carried out from a single sample. As noted above,the ability to remove unnecessary and unhybridized DNA from the reactionmixture significantly aids this process.

[0065] A third advantage to this approach is that following anamplification reaction, the amplicons which have been addressed andbound to a specific site on the array are then available in asite-specific fashion for subsequent analyses, such as by (1) theintroduction of fluorescently labeled nucleotides or (2) thehybridization of oligonucleotides at the end of the reaction bydenaturation of the amplified material followed by hybridization with anappropriate reporter oligonucleotide having specificity for the tetheredamplicon.

[0066] As is described herein, the ability of electronic targeting usedin connection with the combination of an electronically addressablemicrochip and SDA to overcome the above-described limitations of priormethods is demonstrated in two examples of amplicon analysis. First, asdescribed in more detail below, use of a common highly conserved locus(e.g., 16S rRNA) which is shared by numerous species of bacteria may beapplied to multiple comparative analyses of individual samples toidentify different bacteria types. Second, also described in more detailbelow, the electronic microarray of the present invention is used tosimultaneously analyze multiple individual patient samples for thepresence of the human Factor V Leiden (R506Q) gene mutation. The humanFactor V Leiden (R506Q) gene indicates a predisposition to activatedprotein C resistance and venous thrombosis. This example showssuccessful parallel sample analyses from multiple patients. The testmaterial used in this multiple patient sample analysis provides anotheraspect of the present invention, namely, an allele-specificamplification method using SDA, also described in more detail below.

[0067] Other aspects of the present invention are directed to variousnew amplification methods. Such novel SDA methods of the presentinvention are useful for providing amplicons for various analyses. Forexample, some of the SDA methods described herein are useful to optimizeamplification conditions for conducting amplification on anelectronically addressable microchip array. Other SDA methods are usefulto provide amplicons particularly suited for use on an electronicallyaddressable microchip array. Still other SDA methods are useful tooptimize analysis conditions for an analysis conducted on anelectronically addressable microchip array.

[0068] One embodiment of a SDA method of the present invention, morespecifically, comprises an allele-specific SDA method. The methodpreferably selectively amplifies only those strands that include aspecific allele. The method preferably uses amplifying primers designedso their 3′ terminus complements the nucleotide sequence of the desiredallele. The primer may also preferably include a biotin moiety on its 5′end to provide a facile mechanism for capturing the amplicon and/ortarget nucleic acid onto a capture site either prior to amplification orafter amplification following electronic targeting. Additionally, inanother allele-specific embodiment, a method is provided for analyzingmultiple samples containing nucleic acids for the presence of alleles ofa given gene, which comprises amplifying the nucleic acids in eachsample by “two-strand” SDA to produce amplicons, wherein the firstamplification uses primers specific for a first allele and the secondamplification uses primers specific for a second allele, electronicallyaddressing the amplicons on a microarray, hybridizing one or morereporter probes to the bound amplicons, and detecting the presence andlocation of the reporter probes on the microarray.

[0069] In another embodiment of the current invention, a uniquecombination of SDA and simultaneous detection of amplification productson an electronically addressable microchip is provided. In a preferredembodiment, SDA is carried out at the surface of a designated positionon an electronic microchip wherein both upstream and downstream primersnecessary for amplification are anchored to the same discrete capturesite on a microarray. In one such embodiment, the primers are pairedusing a unique branched moiety that is “anchored” to the surface of themicrochip. This branched primer pair design provides closely spacedprimers having a defined distance and location from one another. Thisarrangement further provides a means by which the rate of SDA can becontrolled. Moreover, combined with other elements of the invention,single stranded amplification products being created at the location ofthe primer pair may be easily and quickly addressed and captured byunused branched primer pairs onto the same or adjacent designatedcapture sites on the electronic microchip for further SDA.

[0070] In a preferred embodiment, each primer of the above mentionedprimer pair further includes nucleic acid sequence encoding one strandof an endonuclease restriction site positioned 5′ to a nucleic acidsequence having nucleic acid sequence complementary with a targetmolecule. In a further preferred embodiment, the sequence of therestriction sites in the primers are unmodified in that the nucleic acidbackbone comprises a natural phosphate backbone that is cleavable byaction of the restriction enzyme. Additionally, the restriction sitesuseful in SDA may be any restriction site typically used in SDAprocedures as disclosed in the references incorporated herein such asHincII, HindII, Bso BI, AvaI, Fnu4HI, Tth111I, and NciI. Otherendonucleases can also be used in this method including BstXI, BsmI,BsrI, BsaI, NlaV, NspI, PflMI, HphI, A1wI, FokI, AccI, TthIIII, Mra I,Mwo I, Bsr BI, Bst NI, Bst OI, and Bsm AI. Additionally, the enzyme neednot be thermophilic. Moreover, it is a further preferred embodiment thatthe double stranded SDA amplification product produced during primerextension become hemimethylated or hemiphosphorothiolated (or otherhemimodified form known to those skilled in the art) so that the doublestranded SDA amplification product can be properly “nicked” at theprimer restriction site for normal SDA amplification. For example, thesubstituted deoxynucleosidetriphosphate should be modified such that itwill inhibit cleavage in the strand containing the substituteddeoxynucleotides but will not inhibit cleavage on the other strand.Examples of such substituted deoxynucleosidetriphosphates include2′deoxyadenosine 5′-O-(1-thiotriphosphate), 5-methyldeoxycytidine5′-triphosphate, 2′-deoxyuridine 5′-triphosphate, and7-deaza-2′-deoxyguanosine 5′-triphosphate.

[0071] In an alternative preferred embodiment, a restriction site may beused in the SDA procedure that does not require the nucleic acidbackbone of the restriction site to be modified as described above. Forexample, BstNBI may be used in connnection with its restriction site tonick the nucleic acid as it does not require modification to achievesingle stranded nicks. Instead, BstNBI performs single stranded nicks asa natural activity.

[0072] The nucleic acid segments of the primer pair complementary totarget sequence may be any useful length that will allow hybridizationunder temperature and buffer conditions appropriate for proper functionof SDA on the microchip. Typically, the target sequences of the primerpair have sequence that is complementary with portions of target nucleicacids that are spaced anywhere from 60 to 120 bases upstream ordownstream, as the case may be, from one another. In all cases eachprimer of the primer pair is complementary to different strands (i.e.,the plus strand or the minus strand) of the target sequence.Additionally, where the primer pair is on a branched moiety the spacingbetween the primers on the branched connecting moiety may be adjusted bymolecular spacer elements to optimally enhance the efficiency of the SDAreaction. Such spacer elements may comprise polyethylene glycolpolymers, polyamino acids, or nucleic acids.

[0073] In another preferred embodiment, the spaced primers may beattached to a branched molecular structure (e.g., a ‘Y’ shapedstructure) at their respective 5′ termini. The branched structure isitself then anchored via a free branch of the Y to designated capturepad sites on the microchip. Attachment chemistry to the microchipsurface may be by streptavidin/biotin coupling well known in the art.Alternatively, attachment chemistry may include chemistry comparable tothat disclosed in any of U.S. Pat. Nos. 5,668,258, 5,668,257, 5,677,431,5,648,470, 5,623,055, 5,594,151, and 5,594,111, herein incorporated byreference. In one preferred embodiment, the branched molecules areformed by nucleic acids attached to an amino acid. In another alternateembodiment, the branched molecules are formed by adding differentspacers, such as polyethylene glycol polymers, polyamino acids, ornucleic acids between the nucleic acid primers and a bifunctionallybranched amino acid (e.g. lysine).

[0074] In yet another embodiment, the anchored SDA amplification primersneed not be branched but instead merely anchored individually to thecapture site in close proximity to each other. Attachment chemistry maybe accomplished as described above.

[0075] In another preferred aspect of the invention, amplification oftarget nucleic acids is carried out exclusively at the site of ananchored primer pair thereby avoiding the uncertainties of amplificationrate commonly associated with solution-based amplification.Particularly, as compared with solution-based amplification, theamplification of multiple targets or multiplex amplification is markedlyimproved. It is probable that such improvement is due to the avoidanceof competition between primers and/or avoidance of primer-primerinteractions that may inhibit binding to target sites. Amplification iskept at one location by the combined influence of electronic addressingof target molecules and SDA products to capture pad SDA sites and by thefact that the primers that allow amplification (i.e., the branched orunbranched primer pairs) are anchored to a fixed location.

[0076] In another preferred aspect of the invention, the target nucleicacid is electronically addressed to the specific site on the microchipprior to amplification. This aspect is an advance over passivehybridization technology in several ways. First, since nucleic acids ina sample solution containing target nucleic acid species areelectronically addressed to specific sites on the microchip, the targetmolecules have a preferred advantage of contacting the primer pairdesigned to capture the target molecule. Secondly, in the event singlestranded nucleic acid target molecules must be generated, conditions inthe sample solution that allow for formation of single stranded speciesmust only be accomplished once rather than repeatedly as is normally thecase with PCR and solution-based amplification. Third, the electronicaddressing and annealing of the target species to specific capture siteson the chip may be carried out in low salt conditions, a situation thatis markedly in contrast to classical passive hybridization technology.Low salt conditions (and electronic addressing) enhance thehybridization of single stranded target species to capture primersbecause such conditions help reduce the reannealing of target nucleicacid strands to their respective complementary strands.

[0077] In another preferred embodiment, the anchored SDA methods of thecurrent invention provide improved efficiency because only one targetspecific “bumper” primer is required for annealing to the targetmolecule at a position on the target 5′ to the target annealing positionof one or the other anchored primers. In another embodiment, two bumperprimers may be included (as in traditional SDA) but inclusion of twoprimers is not necessary. Rather, the use of two bumper primers onlyfacilitates initiation of priming from either direction on any one pairof primer capture probes depending upon which of the two strands oftarget nucleic acid are first captured by the branched primer pair.Inclusion of two bumper primers may further enhance the rate of ampliconformation.

[0078] In yet another aspect of this invention, a method ofamplification of a target nucleic acid sequence (and its complementarystrand) in a sample using SDA under elevated pressure is provided. Byelevating the pressure, the efficiency of the amplification is enhancedbecause the specificity of the restriction endonuclease for its targetsequence is increased. The method involves the steps of 1) isolatingnucleic acids suspected of containing the target sequence from a sample,2) generating single stranded fragments of target sequences, 3) adding amixture comprising (a) a nucleic acid polymerase, (b)deoxynucleosidetriphosphates, a phosphorothioated dNTP, endonuclease,and (c) at least one primer which (i) is complementary to a regionsometimes at or along a portion of the target near the 3′ end of atarget fragment, and (ii) further has a sequence at its 5′ end which isa recognition sequence for a restriction endonuclease, and 4) allowingthe mixture to react under elevated pressure for a time sufficient togenerate amplification products. Where the target nucleic acid fragmentscomprise double stranded nucleic acids, the method further comprisesdenaturing the nucleic acid fragments to form single stranded targetsequences. Where the nucleic acids comprise RNA, it is preferable tofirst use reverse transcriptase in order to convert RNA to DNA, however,RNA is specifically included in all embodiments of the invention.

[0079] In a further embodiment, a method of SDA in conjunction with anelectronic microchip is provided wherein the SDA reaction isligation-based. In this embodiment, two sets of primers are used whereinone primer set is designed so that the primers bind to one strand of atarget sequence adjacent to one another while each of the primers of thesecond set are designed to bind to a portion of one of the primers ofthe first primer set while the other of the second primer set iscomplementary to a portion of the other of the first primer set (i.e.,same as the target strand sequence). When this embodiment is used, itwill be apparent that SDA may be accomplished without the involvement ofbumper primers. In a preferred embodiment, one of the two primer setsmay be “anchored” as described herein.

[0080] In another embodiment, a method of ligation-based SDA is providedwhere the method is unassisted by an electronic microchip. In thisembodiment it is not necessary to, inter alia, anchor any primers, whichis a procedure that assists in separating primer sets during multiplexamplification, because the primers are universal—there is no need todirect target sequences to the ‘correct’ primers.

[0081] In a particular embodiment of the ligation-based SDA method, theprobe set designed to anneal to a target sequence must become ligated toform a “ligated probe template” which template is capable of supportingSDA. In a further preferred embodiment, the ligation-based reaction usesa single pair of amplification primers (i.e., the second primer setmentioned above) which are universally applicable to amplification ofall target molecules in a multiplex test providing in turn for decreasednon-target amplification as well as decreased primer competitioninteractions due to the absence of bumper primers.

[0082] In a further preferred embodiment, the ligated probe template ismodified so that it can not be extended from its 3′ end during initialSDA reaction steps. Modifying the relevant ligation probe prevents theformation of a double stranded nucleic acid the 3′ end of which may becleaved by restriction endonuclease due to formation of what would be acleavable restriction site, as explained in more detail below. Thismodification also prevents amplification of ligated probe template thatmay result from the target-sequence-independent ligation of the ligationprobes.

[0083] In another preferred embodiment of the ligation-based SDA method,the pair of probes used to target a nucleic acid of interest and createa ligation probe template are bifunctional in that each probe of thepair contains a target binding sequence and an “amplification primer”binding sequence (i.e., the second primer set mentioned above). Thesequences specific for target binding are chosen so that they arecomplementary to adjacent sequences of target DNA. The portions of theligation probe template primers having nucleic acid sequence used inamplification are chosen so that a single set of amplification primerscan be used for all target species of interest during SDA.

[0084] In a further embodiment, a first amplification primer binds tothe ligated probe template at the 3′ end of the ligated probe templatesuch that there is created two 5′ overhangs. See FIG. 23(a). Doublestranded nucleic acids with 5′ overhangs are normally capable ofsupporting nucleic acid synthesis from the 3′ end of the recessed strandby a DNA polymerase. As is well known in the art, DNA polymerasefunctions by extending the length of one strand of a nucleic acid byincorporating bases to the strand that are complementary to the opposingstrand.

[0085] However, in a further preferred embodiment, nucleic acidsynthesis from the 3′ terminus of the ligated probe template isprevented due to the 3′ terminus having a modification to keep it fromextending. Those in the art understand that this modification may takemany forms including but not limited to: creating a 3′ base mismatchbetween the ligated probe and the amplification primer; using a 3′terminal dideoxy nucleotide; or modifying the chemical moiety present atthe 3′ carbon of the pentose sugar of the nucleic acid backbone by, forexample, replacing the free 3′ hydroxyl group with a phosphate group, abiotin moiety, or by adding other blocking groups which are well knownto those in the art. (See U.S. Pat. Nos. 5,516,663 and 5,573,907 and5,792,607, incorporated herein by reference, discussing various reagentsthat can be used to modify ends of the ligation probes to prevent targetindependent ligation). This modification prevents the formation of adouble stranded nucleic acid which could be improperly “nicked” byendonuclease during the ligation-based amplification process. Thismodification also prevents amplification of ligated probe template thatmay result from the target sequence independent ligation of the ligationprobes and prevents 3′ extension when ligated probe is bound to primer.This modification also allows the ligation and amplification reactionsto proceed without an additional capture step.

[0086] In a further preferred embodiment, the ligation probes aredesigned to include sequences encoding endonuclease restriction sites,such that these sites are located near the 5′ and 3′ ends of the ligatedprobe template. Restriction endonuclease present in the reaction mixturemay nick the double stranded nucleic acid so that SDA may proceed.Nicking of the DNA rather than cleavage occurs because the strandcomplementary to the 5′ end of the ligated probe is synthesized duringSDA using nucleotides that include a modified nucleotide (for exampledATPαS, or dCTPαS).

[0087] In a further embodiment, the amplicons arising fromligation-based SDA may be addressed to capture sites following theirrespective formation (whether their amplification is made to occur bySDA in solution or directly on the capture sites by primers that areaddressed to the capture sites prior to amplification as describedherein).

[0088] In yet another embodiment of the invention, several means bywhich the presence of target nucleic acids in a sample may be detectedare available due to the combined application of the electronicaddressable chip and anchored SDA. For example, in a preferredembodiment, amplicons that are addressed to capture sites may bediscerned directly by fluorescence, i.e., a fluorochrome may be includedin the buffer so that detection is simultaneous with the production ofamplicons. Examples of such fluorescing compounds includeBodipy-derivatives, Cy-derivatives, fluorescein-derivatives, andrhodamine-derivatives all of which are well known in the art.Alternatively, detection of nucleic acids at capture sites may becarried out directly using chemiluminescence orelectrochemiluminescence. Chemiluminescence incorporates the use of anenzyme linked to a reporter oligonucleotide which, when activated withan appropriate substrate, emits a luminescent signal. Examples of suchenzymes include horseradish peroxidase and alkaline phosphatase both ofwhich are well known in the art. Electrochemiluminescence (ECL) is ahighly sensitive process (200 fmol/L) with a dynamic range of over sixorders of magnitude. In this system, reactive species are generated fromstable precursors at the surface of an electrode. These precursors reactwith each other to form the excited state of the label attached to theDNA strand. The excited state decays to the ground state through anormal fluorescence mechanism, emitting a photon having a wavelength of620 nm.

[0089] The amplification products generated using the primers disclosedherein may also be detected by a characteristic size, for examle, onpolyacrylamide or agarose gels stained with ethidium bromide.Alternatively, amplified target sequences may be detected by means of anassay probe, which is an oligonucleotide tagged with a detectable label.In one embodiment, at least one tagged assay probe may be used fordetection of amplified target sequences by hybriization (a detectorprobe), by hybridization and extension as described by Walker, et al.(1992, Nucl. Acids Res. 20:1691-1696) (a detector primer) or byhybridization, extension and conversion to double stranded form asdescribed in EP 0678582 (a signal primer). Preferably, the assay probeis selected to hybridize to a sequence in the target that is between theamplification primers, i.e., it should be an internal assay probe.Alternatively, an amplification primer or the target binding sequencethereof may be used as the assay probe.

[0090] The detectable label of the assay probe is a moiety which can bedetected either directly or indirectly as an indication of the presenceof the target nucleic acid. For direct detection of the label, assayprobes may be tagged with a radioisotope and detected by autoradiographyor tagged with a fluorescent moiety and detected by fluorescence as isknown in the art. Alternatively, the assay probes may be indirectlydetected by tagging with a label that requires additional reagents torender it detectable. Indirectly detectable labels include, for example,chemiluminescent agents, enzymes which produce visible reaction productsand ligands (e.g., haptens, antibodies or antigens) which may bedetected by binding to labeled specific binding partners (e.g.,antibodies or antigen/habpens). Ligands are also useful immobilizing theligand-labeled oligonucleotide (the capture probe) on a solid phase tofacilitate its detection. Particularly useful labels include biotin(detectabel by binding to labeled avidin or streptavidin) and exzymessuch a horseradish peroxidase or alkaline phosphatase (detectable byaddition of enzyme substrates to produce colored reaction products).Methods for adding such labels to, or including such labels in,oligonucleotides are well known in the art and any of these methods aresuitable for use in the present invention.

[0091] Examples of specific detection methods that may be employedinclude a chemiluminescent method in which amplified products aredetected using a biotinylated capture probe and an enzyme-conjugateddetector probe as described in U.S. Pat. No. 5,470,723. Afterhybridization of these two assay probes to different sites in the assayregion of the target sequence (between the binding sites of the twoamplification primers), the complex is captured on a steptavidin-coatedmicrotiter plate by means of the capture probe, and the chemiluminescentsignal is developed and read in a luminometer. As another alternativefor detection of amplification products, a signal primer as described inEP 0678582 may be included in the SDA reaction. In this embodiment,labeled secondary amplification products are generated during SDA in atarget amplidication-dependent manner and may be detected as anindication of target amplification by means of the associated label.

[0092] In another alternative detection method, a target specificprimer, (i.e., a target signal primer which is a primer that is not abumper primer or an anchored primer), designed to anneal to the targetsequence at a position other than at the anchored primer or bumperprimer sites may be included in the amplification step procedure. Thissignal primer may be labeled with a signal molecule that may in turn beused to detect an extension product formed from extension of the signalprimer during SDA. For example, such label may comprise biotin that maybe captured to a microchip location containing streptavidin whichcapture may be detected by presence of a fluorochrome.

[0093] In still another aspect of the invention, use of a signal primerelongation product or amplicon provides for a means by which the molarratio of one target amplicon strand over the other may be produced sothat single stranded amplified species of the target sequence may bemaintained for capture by capture probes located at specific sites onthe microchip. In other words, the signal primer allows “asymmetricSDA”. Moreover, the amplified signal primed amplicons may beelectronically addressed to secondary capture sites which facilitatesfurther reduction in background signal for enhanced detection.

[0094] For commercial convenience, amplification primers for specificdetecion and identification of nucleic acids may be packaged in the formof a kit. Typically, such a kit contains at least one pair ofamplification primers. Reagents for performing a nucleic acidamplification reaction may also be included with the target-specificamplification primers, for example, buffers, additional primers,nucleotide triphosphates, enzymes, etc. The components of the kit arepackaged together in a common container, optionally includinginstructions for performing a specific embodiment of the inventivemethods. Other optional components may also be included in the kit,e.g., an oligonuclotide tagged with a label suitable for use as an assayprobe, and/or reagents or means for detecting the label.

BRIEF DESCRIPTION OF THE DRAWINGS

[0095] The file of this patent contains at least one drawing executed incolor. Copies of this patent with color drawing(s) will be provided bythe Patent and Trademark Office upon request and payment of thenecessary fee.

[0096]FIG. 1A shows a cross-sectional view of an embodiment of thebioelectronic chip of the present invention.

[0097]FIG. 1B shows a perspective view of the bioelectronic chip fromFIG. 1A.

[0098]FIG. 2A shows a schematic representation of a bacterial 16S rRNAgene comprising a divergent region (having a different sequence perbacterial strain) flanked on both sides by conserved regions (having thesame sequence in each bacterial strain). BBs and Bba represent bacterialsense and antisense bumper primers respectively. Bas and Baa representbacterial sense and antisense amplification primers respectively.Identification of the bacterial strains tested are in the sequencelisting.

[0099]FIG. 2B shows the results of 16S rRNA encoding SDA amplificationproducts resolved on a 1% agarose gel stained with ethidium bromideshowing specific amplification of the divergent regions from eachstrain.

[0100]FIG. 2C shows one aspect of a sandwich assay format used fornucleic acid hybridization on microarrays of the present inventionwherein the assay format utilizes a universal capture probe and asequence specific reporter.

[0101]FIG. 2D shows a sandwich assay format used for nucleic acidhybridization on microarrays of the present invention wherein the assayformat utilizes a sequence specific capture probe and a universalreporter.

[0102]FIG. 3A shows Salmonella-specific BTR labeled reporter used forpassive hybridization of SDA amplicons on a microarray wherein thecapture sites of the microarray include as a control for non-specificbinding of the reporter oligonucleotide to the capture probes orpermeation layer itself a site containing capture probes but no target(+C/−T) and a site containing no capture probe or target (C−/T−).

[0103]FIG. 3B shows a comparison of the relative fluorescence observedfor each bacteria when SDA amplicons were generated and electronicallyaddressed to individual sites on a microarray using universal captureprobes, and sequence-specific btr-labeled reporter probes (designed inthe divergent region of the 16S rRNA gene) were passively hybridized todiscriminate various bacterial strains.

[0104]FIG. 3C shows a comparison of the relative fluorescence observedfor each bacteria when SDA amplicons were generated and electronicallyaddressed to individual sites on a microarray using sequence-specificcapture probes, and universal btr-labeled reporter probes (designed inthe conserved region of the 16S rRNA gene) were passively hybridized tothe captured material.

[0105]FIG. 4A shows a polyacrylamide gel analysis of the allele-specificreactions from five patient samples analyzing for Factor V Leidenmutation in each wherein each genomic DNA sample was amplified twicewith allele-specific SDA using either the normal genotype (Factor VR506), W, or the Leiden mutation (Factor V Q506), M.

[0106]FIG. 4B shows a histogram comparing the fluorescence present ateach addressed site on the array of the allele-specific reactions fromthree of the five patient samples of FIG. 4A.

[0107]FIG. 5A shows a diagram of a first scheme of incorporating afluorescent species in an amplification reaction for detection purposes.

[0108]FIG. 5B shows a diagram of a second scheme of incorporating afluorescent species in an amplification reaction for detection purposes.

[0109]FIG. 6A shows a fluoroscopic analysis of a microchip where the SDAtemplate was absent as a control.

[0110]FIG. 6B shows a fluoroscopic analysis of a microchip where BsoBIwas not included in the reaction as a control.

[0111]FIG. 6C shows a fluoroscopic analysis of a microchip where the SDAtemplate was passively hybridized overnight.

[0112]FIG. 7 shows the Mean Fluorescence Image of the fluoroscopicanalysis of FIGS. 6A-6C.

[0113]FIG. 8 shows a fluoroscopic analysis of a microchip where the SDAtemplate was electronically targeted.

[0114]FIG. 9 shows the titration of Factor V PCR in the SDA template ofFIG. 8.

[0115]FIG. 10(a) shows the gel product of a NASBA amplification.

[0116]FIG. 10(b) shows fluoroscopic analysis of a sandwich assay resultof NASBA Tax plasmid after electronic targeting to a microarray.

[0117]FIG. 11 shows a graph of the titration of non-cleavable SDAprimers in Factor V anchored SDA.

[0118]FIG. 12 is a schematic diagram of the anchored primers showingaspects of the branched primer design.

[0119]FIG. 13 is a schematic diagram showing the stepwise process ofcreating amplicons from target nucleic acid sequence at a branchedprimer pair site.

[0120]FIG. 14 is a schematic diagram showing the nature of using asignal primer to generate asymmetric ratios of nucleic acid ampliconchains such that the amplicons with signal may be electronicallyaddressed to a capture pad for signal detection.

[0121]FIG. 15 is a schematic diagram showing anchored non-branched SDAtarget primers.

[0122]FIG. 16 is a diagram showing the layout of a microchip pad withthe locations on the pad to which the various target species tested havebeen addressed as explained in Example 7.

[0123]FIG. 17 is a photographic image of a control SDA reaction whereinno target nucleic acid was present.

[0124]FIG. 18 is a photographic image showing specific localization ofSDA amplified Factor V target in the presence of multiple target specieson only SDA capture primer pairs specific for Factor V which had beenpreviously addressed to only the four capture sites.

[0125]FIG. 19 is a photographic image showing specific localization ofSDA amplified Factor V and Chlamydia targets which were amplified in thepresence of multiple target species and SDA capture primer pairsspecific for Factor V and Chlamydia that had been previously addressedto specific capture sites.

[0126]FIG. 20 is a photographic image showing specific localization ofSDA amplified Factor V, Chlamydia, and Hemachromatosis gene targetswhich were amplified in the presence of multiple target species and SDAcapture primer pairs specific for Factor V, Chlamydia, andHemachromatosis that had been previously addressed to specific capturesites.

[0127]FIG. 21 is a PAGE gel showing results of a multiplex solutionbased SDA reaction for Factor V, Chlamydia, and Hemachromatosis genetargets. The minus lane indicates no template DNA present, while theplus lane indicates addition of template DNA.

[0128]FIG. 22 is a diagram showing a proposed reaction sequence forsynthesis of a branched SDA primer pair.

[0129] FIGS. 23(a-c) illustrate a reaction pathway for theligation-dependent amplification of a target nucleic acid sequence.

[0130]FIG. 23(d) illustrates the ligation probes and amplificationprimers that would be used to detect the Salmonella spaQ gene present ina sample using the method illustrated in FIGS. 23(a-c).

[0131]FIG. 24 is a graph showing specific amplification using theexonuclease ligation dependent SDA aspect of this invention, asexplained in Example 10, in conjunction with a microelectrode arrayhaving capture probes for five bacterial genes pre-arranged at discretelocations.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0132] The present invention relates broadly to devices, methods, andcompositions of matter for amplifying nucleic acid sequences in a sampleand for analyzing those sequences. The amplification and the analysisare optimally accomplished using SDA and bioelectronic microchiptechnologies.

EXAMPLE 1

[0133] In a preferred embodiment of this invention, a microchip devicecomprising an electronically controlled microelectrode array is providedfor the analysis of target nucleic acids of interest. In contrast to theuniform hybridization reaction environment and passive hybridizationused in other microchip devices, the electronic microchip-based devicesof the present invention offer the ability to actively transport andhybridize target and/or primer nucleic acids to capture probes atdiscrete locations on the surface of the microelectrode array.

[0134] Referring now to FIGS. 1A and 1B, a simplified version of theelectronically addressable microchip-based hybridization system embodiedwithin this invention is illustrated. Generally, a substrate 10 supportsa matrix or array of electronically addressable micro-locations 12 whichmay be any geometric shape such as square or circular. For ease ofexplanation, the various micro-locations in FIG. 1A have been labeled12A, 12B, 12C and 12D. A permeation layer 14 is disposed above theelectrodes 12 and may extend over the entire device surface. Thepermeation layer 14 permits transport of relatively small chargedentities through it, but limits the mobility of large charged entities,such as nucleic acids, to keep the large charged entities from easilydirectly contacting the electrodes 12 that are located under thepermeation layer of a capture site. The permeation layer 14 also reducesthe electrochemical degradation that could occur if direct contact weremade with the electrodes 12. Electrochemical degradation is sometimesinduced by both formation of reactive radical species and extreme pH atthe electrode surface during the electrolytic reaction. The permeationlayer further serves to minimize the strong, non-specific adsorption ofnucleic acids to electrode surfaces. Attachment regions or capture sites16 are disposed upon the permeation layer 14 and provide for specificbinding sites for target materials. The capture sites 16 in FIG. 1A havebeen labeled 16A, 16B, 16C and 16D to correspond with the identificationof the electrodes 12A-D, respectively.

[0135] The central area of the microchip contains reservoir 18 forplacing sample nucleic acids above the area containing the multiplicityof capture sites 16. In a preferred embodiment, charged molecules 20,such as charged target or probe nucleic acids located within reservoir18 may be transported to any of the specific micro-locations 12. Whenactivated, a micro-location 12 generates the free field electrophoretictransport of any charged molecule 20 (e.g., probe, target nucleic acidsor amplicons) toward the electrode 12A. As a further example, addressingelectrode 12A with a positive bias and electrode 12D with a negativebias, causes electrophoretic lines of force 22 to run between electrodes12A and 12D and further cause the transport of charged molecules 20having a net negative charge toward the positive electrode 12A. Chargedmaterials 20 having a net positive charge move under the electrophoreticforce toward the negatively charged electrode 12D. When the netnegatively charged molecules 20 contact the capture sites 16A permeationlayer as a result of its movement under the electrophoretic force, thecharged molecule 20 becomes attached to the capture sites attachmentlayer 16A. Attachment may be by many methods as discussed belowincluding attachment by hybridization of a target charged molecule 20 toa complementary nucleic acid probe that is anchored to the capture site16.

[0136] Electronically addressable microchip arrays of the presentinvention overcome the size limitations of capture probeoligonucleotides and complexity requirements of passive microchipdevices. The addressable microchip also greatly reduces the need forstrand separation, at least in part, because of the use in the currentsystem of a low ionic environment which inhibits the formation of doublestranded nucleic acid that is in solution prior to capture andamplification of the nucleic acid at a capture site. In addition, themicrochip arrays of the present invention allow multiple independentsample analyses (i.e., multiplex sample analysis) upon the same openmicroarray surface by selectively and independently targeting differentnucleic acid samples to various microelectrode locations. In otherwords, they allow parallel multiple sample processing on an open array.As is described in detail below, the capability of electronic targetingto overcome the above-described limitations of passive hybridizationmethods is demonstrated in the following two examples A and B.

EXAMPLE A Parallel Analysis of Single Target Nucleic Acids in a Sample

[0137] In a first example, a parallel analysis of the capture anddetection of a single nucleic acid in a test sample was performed usinga common locus (16S rRNA) shared by different bacterial species.Multiple comparative analyses of individual samples were used toidentify different bacteria types.

[0138] The secondary structural requirements of the 16S ribosomal RNAsubunit demands highly conserved nucleic acid sequences in the 16S rRNAgene. Thus, there is limited sequence divergence in this gene betweendifferent species of bacteria. Despite the overall high sequenceconservation, there are pockets of microheterogeneities within the 16SrRNA gene, which can be exploited to discriminate between closelyrelated bacterial species. See, e.g., C. Woese, 51 Microbiol. Revs.221-271 (1987).

[0139] The bracketing of these microheterogeneities by conservedsequences provides opportunities to design many primers for consensusamplification (i.e. uniform amplification using the same primersregardless of species) for almost all bacterial species containing theconserved sequences. As shown in FIG. 2A, SDA primers were designed inthe conserved regions that flank the polymorphic region and used in SDAreactions. The resulting amplicons included the various sequences of the“microheterogeneity domains” of the 16S rRNA genes. These were analyzedby a variety of methods.

[0140] As demonstrated below, consensus SDA primers can be used for thegeneration of species-specific amplicons which in turn can be readilyanalyzed by hybridization on active microelectronic arrays. Similarstudies have been reported using PCR as a means of target amplification.See, e.g., D. Linton, et al., 35 J Clin. Microbiol. 2568-72 (1997), M.Hughes, et al., 35 J. Clin. Microbiol. 2464-71 (1997). However, thepresent invention uses a sandwich assay in which a single-strandedcapture probe is electronically deposited on the array, and serves tocapture one strand of a charged molecule such as a target nucleic acidor amplicon thereof. In a preferred embodiment, a multiplicity ofmolecules such as nucleic acid capture probes can be electronicallydeposited on different pads of the array. Following capture of thecharged molecule to the capture sites, the captured molecule may bedetected by a labeled reporter probe that binds to the capturedmolecule.

[0141] As is shown schematically in FIG. 2A, the 16S rRNA gene near its3′ end has an oligonucleotide region stretching greater than twentycontiguous nucleotides of polymorphic sequence 24 flanked on both sidesby conserved sequences 26. The unique sequences 24 of each bacterialspecies specified in the sequence listing herein were used in an SDAreaction in the electronically addressable microchip to show that it ispossible to discriminate between different bacterial species bycapturing these polymorphic sequences and their respective amplicons atspecific capture sites. More particularly, primers were designed havingnucleic acid sequence complementary to the highly conserved loop IIIstructure of the small subunit of the bacterial ribosomal RNA 26. A 3′base complementary to a species-specific allele or point mutation in thesequence were also designed and made. As shown in FIG. 2A, this primerconfiguration facilitates design of both SDA amplifier and bumperprimers for any particular group of organisms having the same conservednucleic acid sequences. Primers can also be made so that they are“universal” for use in SDA to detect organisms of a group.

[0142] In a specific example, genomic DNA from bacteria (E. coli0157:H7, Salmonella typhimurium, Shigella dysenteriae, and/orCampylobacter jejuni) were amplified. The same set of 16S rRNA encoding“consensus” primers (described in more detail below) were employed ineach SDA reaction. The products of the SDA reactions were resolved on a2% agarose gel to compare the amplification efficiencies betweendifferent bacterial species. The resulting gel is shown in FIG. 2Bwherein similar levels of amplification efficiency were obtained foreach of E. coli 0157:H7, Salmonella typhimurium, and Shigelladysenteriae, and in other experiments utilizing genomic DNA fromCampylobacter jejuni (data not shown). Table I, below, shows theoligonucleotide sequences used for amplification and microarray analysisof these bacterial species.

[0143] Two different approaches were used to analyze the amplificationproducts. A first analysis approach used a common or universal captureprobe and a sequence specific reporter (i.e. a universalcapture/specific reporter method). A second analysis approach useddiscriminating capture primers and a universal reporter (i.e. a specificcapture/universal reporter method). As is shown in FIGS. 2C and 2D,universal capture probes 28 and universal reporters 32 were designed tospan at least a portion of one of the conserved regions 26 (FIG. 2A) ofthe gene. As is also shown in FIGS. 2C and 2D, sequence specific captureprobes 35 and sequence specific reporters 34 were designed to span atleast a portion of the polymorphic region 24 (FIG. 2A).

[0144] Where universal capture probe 28 was used to capture nucleicacids, the initial step of hybridization between a target nucleic acidand a universal capture probe was performed electronically for severalreasons. First, electronic hybridization greatly accelerates thekinetics of hybridization which is important when working with lowconcentrations of material, such as a highly diluted target or amplicon.Second, because of the extremely low ionic strength of the buffersystems used, targets and amplicons remain single stranded facilitatingcapture by probes and much less competition from the complementarystrand of target or amplicon and, hence, higher net specific binding ofthe nucleic acid to the capture probe. Consequently, electronichybridization allows a much higher level of nucleic acids hybridizing atthe site of the capture probe resulting in greater detection anddiscrimination sensitivity.

[0145] In each case of this example, reporter hybridization was passive,i.e. performed at elevated salt and temperature without the aid ofelectronics, although electronics could be used. In this particularexample, since the concentration of the single stranded labeledoligonucleotides was so high, there was little practical kineticadvantage to be obtained through the use of electronic hybridizationconditions. However, under different circumstances, the use ofelectronics during reporter hybridization may be beneficial.

[0146] As shown in FIG. 3A, amplicons were addressed to the capturesites on the microchip and detected by a fluorescent reporter molecule(as described below). The relative fluorescence on capture sites towhich were hybridized amplification products of bacterial 16S rRNAtargets discussed in FIG. 2A were highly discriminated (i.e., apolymorphism specific Salmonella reporter, a polymorphism Shigellareporter, and a polymorphism Campylobacter reporter). In theseexperiments, universal capture probes (“S”) were first addressed to themicrochip along with a non-specific capture probe (“NS”) as a control.Amplicons from each strain-specific SDA reaction were then addressed toeach corresponding row and passively hybridized with a specific reporterprobe. FIG. 3A shows results for Salmonella-specific reporter. As acontrol for non-specific binding of the reporter probe to the permeationlayer, a minus capture/minus target control was also performed (−C/−T).As shown, only the Salmonella amplicon addressed capture sites gave apositive signal. As shown in FIG. 3B, not only were high discriminationratios obtained for Salmonella as shown in FIG. 3A, high discriminationratios were also seen between the various other bacterial targets.(fluorescent imaging data not shown.)

[0147] Where sequence specific capture probes 35 were used to capturenucleic acids, the initial step of hybridization between target andcapture probe was also performed electronically. As in the universalcapture example above, The reporter sequence was designed to recognize aconserved region of the 16S rDNA amplicons 26. As shown in FIG. 3C, thisapproach provided even higher discrimination ratios between the matchand the mismatch.

EXAMPLE B Simultaneous Analysis of Multiple Target Nucleic Acids

[0148] In a second example, multiplex amplicon analysis was performed onthe electronic microarray of the present invention. In this example,target nucleic acids from multiple patient samples were sequentiallyaddressed to capture sites in order to detect the presence of the humanFactor V Leiden (R506Q) gene (which indicates a predisposition toactivated protein C resistance and venous thrombosis). In this example,capture probes were designed so as to be specific for alleles of theR506Q gene thereby providing a method to detect allele-specific SDA.

[0149] As explained herein, since each capture site on the openmicroarray may be individually electronically controlled, multiplesamples may be analyzed. Following amplification and position-specifictargeting of each sample amplification reaction, the array was evaluatedin a site-specific fashion for the presence or absence of targetedamplicons. The test system examined the presence or absence of the humanFactor V Leiden mutation in several blood samples. See, X. Liu, et al.,4 Mol. Pathol. 191-197 (1995). The Leiden mutation is a single pointmutation at the protein C cleavage site of the Factor V gene. Where thismutation has a homozygous presence in a patient, it leads to activatedprotein C resistance and a predisposition to deep venous thrombosis.See, e.g., R. Bertina, et al., 369 Nature 64-67 (1994).

[0150] To aid in discrimination, an allele-specific SDA assay wasdeveloped. The allele-specific SDA was designed to selectively amplifyeither the normal or the mutant Factor V Leiden allele. The SDAamplifying primers in the antisense orientation were designed with their3′ termini complementary to either the normal nucleotide base G, or theLeiden point mutation nucleotide base A, present in the sense strand ofexon 10. Table I, below, shows the oligonucleotides used foramplification and microarray analysis of the Factor V gene. Thecorresponding sense primer was common in all reactions. However, thesense primer was modified by incorporating a biotin moiety on its 5′ endin order to provide a facile mechanism for capturing any amplicons onthe array following electronic targeting. TABLE I Oligonucleotides Usedfor Amplification and Microarray Analysis Bacterial 16S Sequence(5′-3′)¹ Position² BBs CAAATGAATTGACGGGGGCC (SEQ ID. NO. 1) 927-946 BbaAAGGGTTGCGCTCGT (SEQ ID. NO. 2) 1134-1120 Bas ACCGCATCGAATGCATGTCCTCGGGTGCATGTGGTTTAAT (SEQ ID NO. 3) 961-975 BaaACGATTCAGCTCCAGACTTCTCGGGTAACATTTCACAACAC (SEQ ID NO. 4) 1114-1090 Brecoli btr-CTCATCTCTGAAAACTTC (SEQ. ID. NO. 11) Brsdysbtr-CGTATCTCTACAAGGTTC (SEQ. ID. NO. 12) Brstyp btr-TCCATCTCTGGATTCTTC(SEQ. ID. NO. 13) Brcjej btr-CATATCTCTATAAGGTTC (SEQ. ID. NO. 14) HumanFactor V Sequence (5′-3′)¹ Position² FVBs ACTACAGTGACGTGGACATC (SEQ ID.NO. 5) FVBa TGTTATCACACTGGTGCTAA3 (SEQ ID. NO. 6) FVAs bio- (SEQ ID. NO.7) ACCGCATCGAATGCATGTCCTCGGGTCTC TGGGCTAATAGGA FVA wtACGATTCAGCTCCAGACTTCTCGGGTAAT (SEQ ID. NO. 8) ACCTGTATTCCTC EVA mACGATTCAGCTCCAGACTTCTCGGGTAAT (SEQ ID. NO. 9) ACCTGTATTCCTT FVRbtr-CTGTATTCCTCGCCTGTC (SEQ.ID. NO. 10) # and antisense bumper primersfor Factor V.

[0151] In this multiplex Factor V gene study, four clinical DNA sampleswere analyzed in duplicate without prior knowledge of the patient'sFactor V Leiden mutation status. Two allele-specific SDA reactions wereconducted per sample (containing either normal or mutant primers) toexamine each patient's genotype. The amplifications were conducted inparallel. The PAGE results from five of these pair-wise reactions isshown in FIG. 4A wherein the allele-specific amplification reactionsunder these conditions are shown to be highly specific. That is, theselective absence of visible mutant or normal-type amplicons indicatesthat the amplification reaction is sensitive to the presence or absenceof the Factor V Leiden mutation in these individuals.

[0152] All amplicon reactions, regardless of the presence or absence ofamplified material as determined by gel analysis, were uniformly treatedand sequentially targeted to specific locations upon the microarray.Representative results from three DNA patient samples are shown in FIG.4B. These samples were targeted in duplicate. The presence or absence ofa fluorescent signal from a hybridized reporter oligonucleotidecomplementary to a conserved region on the target amplicon (i.e., a“universal” reporter probe) indicates the presence or absence of FactorV amplicons. As can be seen, the fluorescent signal correlates well withthe gel results shown in FIG. 4A.

[0153] As is shown in FIG. 4A, positive signals were several foldgreater than background signals. In general, the true mutant signal waslower than that from wild type amplicons (as shown in FIG. 4B). Thesites were scored simply by making the criteria for a positive signal tobe at least twofold above the background fluorescence present atnon-addressed capture sites. As shown in Table II, below, there wascomplete correlation between the presence of amplified material by gelanalysis and the presence of strong or moderate fluorescent signals uponthe array.

[0154] The strength of the fluorescent signal approximated the apparentquantity of amplified material. This was most striking in those sampleswith an apparently less efficient amplification reaction such as wasseen with the DNA from patient 961 a in FIGS. 4A and 4B. In short, theseresults show that multiple sample analysis by the serial application ofsamples followed by single reporter detection works using amicroelectronic array, and shows that this process may serve tosupplement or replace other forms of analysis, e.g. gel electrophoresis,in the same or similar analyses.

[0155] Since these samples were analyzed prior to knowledge of theirmutational status, it was of interest to determine whether the apparentallele specificity of the amplification reaction did, in fact,correspond with clinical status. As is shown in Table II, below, theselectivity of the allele-specific amplification reaction was incomplete agreement with the Factor V Leiden mutational status of eachsample as determined by PCR and MnlI restriction site analysis. (R.Press, unpublished observations). Thus, combined with allele-specificSDA, analysis of amplicon product formation upon an electronicallyaddressable array is a useful method for detecting genetic pointmutations in mupltiple patient samples. TABLE II Allele Specific FactorV SDA Amplification Results PAGE¹ Microarray¹ Patient Sample Date wt mutwt mut Genotype² 951961 04/10/97 X X X X Heterozygous 951961 06/04/97 XX X X Heterozygous 952018 04/10/97 X O X O Homozygous wt 952018 06/04/97X O X O Homozygous wt 960286 04/10/97 O X O X Homozygous mut 96028606/04/97 O X O X Homozygous mut

[0156] Experimental Protocol used in the Above Described Data

[0157] Materials—Deoxynucleoside 5′-triphosphates (dGTP, dATP, TTP) werepurchased from Pharmacia, Alameda, Calif. 2′-deoxycytosine5′-O-(1-thiophosphate) (dCTPαS), BsoB1 restriction endonuclease and Bstpolymerase were supplied by Becton Dickinson, Sparks, Md.Oligonucleotides were synthesized by Oligos, Etc., Wilsonville, Oreg.

[0158] SDA Amplification—Amplification reactions utilized either 1 μg ofgenomic DNA (16S) or 0.1 μg of genomic DNA (Factor V) in a volume of 30μl. Amplification conditions and concentrations were adapted from thatpresented previously (see, C. Spargo, et al., 10 Molecular and CellularProbes 247-256 (1996)) with the following changes: The 5'to 3′exonuclease deficient polymerase Bst replaced the exo-BCA polymerase, asdisclosed and used in M. A. Milla et al., Biotechniques, v24, p 392-396,March 1998 herein incorporated by reference. For 16S amplification,25U/reaction (Bst) and 60U/reaction (BsoB1) were used. Oligonucleotidesemployed for amplification reactions are shown in Table I above.Reactions were allowed to proceed for 30 minutes at 60° C. and thenterminated by the addition of 10 μL of 100 mM EDTA and then stored at−20° C.

[0159] Gel Electrophoresis—Amplification reactions were analyzed usingstandard protocols with either 1% agarose gel or with 6% polyacrylamidemini gels (Novex, San Diego, Calif.) followed by ethidium bromidestaining. Images were obtained using an Alphalnotech Chemimager (SanLeandro, Calif.).

[0160] Electronic Microarray Analysis—The microelectronic array assemblyhas been described previously. See, R. Sosnowski, et al., 94 J. Poc.Natl. Acad. Sci. USA 119-123 (1997). Electronic targeting of captureoligonucleotides (biotin-GGATGTCAAGACCAGGTAAGGTTCTTC, Genbank locus988-1014 bp (SEQ ID NO. 15) and hybridization of amplicons (16S) orreporter oligonucleotide (Factor V) utilized conditions reportedelsewhere. See, R. Sosnowski, supra, and C. Edman, et al., 25 J. NucleicAcids Res. 4907-4914 (1997). In brief, crude amplification reactionswere either spun for two minutes through G6 columns (Biorad, Hercules,Calif.) preequilibrated with distilled water or dialyzed in multiwellplates (Millipore, Bedford, Mass.) for more than or about five hoursagainst distilled water. The prepared samples were then mixed in a 1:1ratio with 100 mM histidine and heated at 95° C. for five minutes priorto electronic addressing. For analysis of 16S amplicons, electronichybridization of the amplicons was performed, followed by hybridizationin 6× SSC of a fluorescent labeled oligonucleotide reporter homologousto a specific bacterial sequence. Specific nucleotide sequences areshown in Table I, above. Passive hybridization was allowed to proceedfor 30 minutes at room temperature. The microchips were washed 5 to 8times using 0.1× STE/1% SDS followed by 1× STE. Similar conditions wereemployed for the single target experiment above using the 16S bacterialrRNA sequence-specific Biotin-captures and a common btr-labeled reporterfor detection. For analysis of Factor V amplicons, a fluorescent-labeledoligonucleotide (btr-CTGTATTCCTCGCCTGTC (SEQ ID NO. 10) was introducedin 6× SSC and allowed to hybridize for 30 minutes at room temperature.The array was then washed in 0.1× STE/1% SDS followed by 1× STE.

EXAMPLE 2

[0161] Turning now to the electronic amplification aspect of the presentinvention, target nucleic acid is electronically concentrated in thevicinity of anchored primers located on a capture site and used in anSDA or other amplification method. The target nucleic acid may beelectronically concentrated and hybridized to binding molecules (e.g.,capture probes) on the surface of the microchip capture sites prior tothe introduction of SDA reaction components (i.e. enzymes, nucleotides,etc.) thereby increasing the efficiency and decreasing the timenecessary for hybridization of target nucleic acid to the anchoredcapture primer on the capture site. Hybridizing the target nucleic acidto specific locations on the microarray prior to addition of SDAreaction components also permits the array surface to be washed toremove unwanted and possibly interfering non-target nucleic acids fromthe reaction environment. Thus, amplification reactions, such asanchored SDA, can benefit greatly by using an electronically addressablemicroarray system.

[0162] The components of the amplification reaction itself (withouttemplate and amplification primers) are introduced and the amplificationreaction allowed to proceed. There are at least three advantages toemploying electronic targeting of template molecules. The first is thatthe overall time and efficiency of the amplification process isdramatically improved since a major rate-limiting step (that of the timerequired for the template to find the anchored primers) is removed fromthe overall reaction rate. Also, the use of the electronic concentrationand hybridization increases the number of target molecules at theselected site, as compared to non-electronic passive hybridization foran equivalent time period, thereby increasing the absolute numbers ofstarting template molecules for amplification resulting in improvementin both the overall yield of the amplification process and thesensitivity of the system to lower starting template numbers.

[0163] The second advantage is that discrete target nucleic acid samplescan be applied to specific locations upon the array surface therebyallowing multiple and different nucleic acids to be amplifiedsimultaneously on one array. Alternatively, a nucleic acid may betargeted to several different locations, each containing specific setsof amplification primers so that multiple different amplificationreactions can be simultaneously carried out from a single sample. Asnoted above, the ability to remove unnecessary or unhybridized nucleicacids from the reaction mixture significantly aids this process.

[0164] A third advantage to this approach is that following theamplification reaction, the captured amplicons are available in asite-specific fashion for subsequent analyses, either by introduction offluorescently labeled nucleotides or by the incorporation of labeledoligonucleotides during the course of the amplification reaction or byhybridization with an appropriate reporter oligonucleotide at the end ofthe reaction by denaturation of the amplicons that are bound to thecapture sites.

[0165] In an example of this electronic addressing embodiment, anexperimental protocol was designed to enhance anchored Factor V SDAsensitivity by using electronic hybridization of Factor V encodingtemplate nucleic acid to anchored SDA primers (Seq. I.D. Nos. 20 and 21)on a microchip array. The SDA primers were biotinylated at theirrespective 5′ ends. These primers also contained a BsoBI enzyme cleavagesite. The reaction mix included the bumper primers (Seq. I.D. Nos. 22and 23) for SDA. The microchip array was prepared by scraping thestreptavidin-agarose layer from the outer electrodes of the microchip.The edges of the chip were waterproofed with Rain-X and the surface wasbuffed clean with a cotton swab applicator. The array was incubated withmilli-Q water for at least 30 minutes at room temperature.

[0166] Solutions were prepared for electronic addressing on themicrochip. SDA primers in 1 μM in 50 mM histidine buffer, 1 μMbiotinylated T12-btr oligonucleotide in 50 mM histidine buffer, and 50mM histidine wash buffer were prepared. The microchips were washed with50 mM histidine buffer, and biotinylated T12-btr oligonucleotides wereaddressed using a standard A/C protocol (800 nAmps for 25 seconds) toselected capture sites to check the quality of the streptavidinmicrochips. The SDA primers were addressed to selected capture sites asshown using the standard A/C protocol.

[0167] For electronic hybridization (as opposed to passivehybridization) experiments, double stranded PCR nucleic acid templateswere first denatured at 95° C., and an equal volume of 100 mM histidinebuffer was added to the template. The template mixture was thenelectronically hybridized to the capture SDA primers using a standardA/C protocol for hybridization (1.6 μAmps, 60 seconds).

[0168] For passive hybridization experiments, asymmetric PCR nucleicacid templates were first denatured at 95° C. for 5 minutes. Thesolution was then brought to a 4× SSC concentration with a 20× SSC (3MNaCl, 0.3 M NaCitrate) stock and 20 μl of the mixture was pipetted ontoa microchip (which had been previously electronically addressed with SDAprimers) and incubated at room temperature overnight.

[0169] After incubation the microchip arrays were washed 2× with waterand incubated with 1 mg/ml BSA for 30 minutes at room temperature toblock any non-specific binding sites. The microchips were washed againwith water (2×) and pre-warmed at 60° C. for 5 minutes. All SDAsolutions were also pre-warmed at 60° C. for 5 minutes. Afterpre-warming, the water was removed from the microchips and incubatedwith 10 μl SDA reaction mix (40 mM K₂HPO₄ pH 7.6, 1.6 mM each dCTPαS,dTTP, dATP and dGTP, 8.3 mM MgCl₂, 1.3 units BsoBI and 0.5 units Bstpolymerase) for 30 minutes at 60° C. in a humidifying chamber. Thereaction was stopped by removing the supernatant from the microchipsurface to an eppendorf tube containing 2 μl of 100 mM EDTA.

[0170] After the SDA reaction, the microchips were washed 3× with 0.5×SSC, pH 7.2. The SDA products were then denatured on the microchip insitu with addition of 0.5× SSC, pH 12.0 for 4 minutes, washing themicrochip with additional buffer after every minute. The microchips werethen washed with 0.5× SSC, pH 7.2 at least 3 times, then with 4× SSC, pH7.2 at least three times. The microchips were incubated with a 1 μM mixof btr-labeled reporter oligonucleotides (such as Seq. I.D. Nos. 24 or44) in 4× SSC for 3 minutes at room temperature, washed extensively with4× SSC at room temperature, then imaged.

[0171] For passive hybridization of Factor V template on microchipsaddressed with Factor V SDA primers at distinct sites, microchips wereaddressed with 1 μM of either Factor V SDA primers, or Factor V SDAprimers lacking a BsoBI site as a negative control for the SDA reaction.Since the negative control lacks a BsoBI site, the reaction can onlyundergo primer extension upon binding of a template and not SDAamplification. This reaction controls for the presence of non-specificbinding as well as the production of non-specific amplification productswith which the reporter oligonucleotides may react. A no-templatecontrol was also present. These microchips were then fluoroscopicallyanalyzed for Factor V amplicons having the fluoroscopically labeledbtr-reporter oligonucleotides. SDA products were seen only in themicrochip where SDA template was passively hybridized overnight (FIG.6C). No products were seen in the no-template control microchip (FIG.6A), or in the microchip (FIG. 6B) where BsoBI was not included into thereaction (another negative control for the SDA reaction). In themicrochip that was passively hybridized (FIG. 6C), the SDA products areseen only in the area where the SDA primers were addressed, not in thenon-cleavable SDA primer quadrant of the array, again confirming thatthe product detected is specific and is driven by an SDA-based process.The drawback of this assay is that the images seen after the SDAreaction were very weak, having MFI (Mean Fluorescence Image) values of14 at an integration time of Is for non-diluted template levels (FIG.7).

[0172] For electronic hybridization of Factor V template to anchored SDAprimers on a microchip, experiments were conducted in a manner parallelto that carried out for passive hybridization, with the exception thathybridization of the template was facilitated by electronic addressing.Additionally, the template was also serially diluted. As a control,passive hybridization of the factor V template was carried out andresulted in a very small increase (approximately 1 MFI unit) overbackground in the SDA reaction. Again, no signal was seen in thenon-cleavable primer quadrant, indicating the need for SDA-directedamplification in this system. In contrast, the microchip that waselectronically hybridized showed a signal in the SDA primer quadrant(FIG. 8) and showed a significant signal in all dilutions tested (FIG.9). Even at a dilution of 1:100 of the Factor V template, the signal wasstill very high, at approximately 19.4 MFI/sec. Given that the MFIsignal of a 1:100 dilution of the electronically addressable microchipwas 19.4 times higher than the signal from a passively hybridized chipin this experiment, (and 1.4 times higher than in the passivehybridization experiment, above, where the template was not diluted) theefficiency of the SDA assay increased approximately 140-1940 percent byusing an electronic hybridization protocol. This demonstrates thatelectronic hybridization of the template to SDA primers anchored on themicrochip increases the sensitivity of the assay approximately 1000fold. In addition, the time required to perform the entire SDAexperiment was reduced by one full working day (as compared to passivehybridization wherein the template needed to be incubated overnight toachieve efficient binding levels).

[0173] In another example, we show that electronic addressing of targetmolecules to capture sites facilitates the amplification of DNA or RNAtarget nucleic acids using the technique known as nucleic acidsequence-based amplification (NASBA). In this method three differentenzymatic activities are used in a coordinated fashion with anisothermal method of amplification. In this electronically-mediatedprocess, the simultaneous or multiplex amplification of differentsequences is possible either by site specific targeting andamplification or by using multiple primer sets. Moreover, NASBA, aspracticed in the invention, may use either anchored or solution-basedprimers in the amplification reaction. In either case, the reaction isenhanced using electronic addressing of the target to its respectiveamplification primers.

[0174] In this example, target nucleic acid sequences were firstelectronically hybridized to discrete locations upon a microchip.Unwanted or non-specifically binding nucleic acids were removed eitherby electronic washing, or passive (non-electronic) washing or by acombination of the two. Following the wash step, the hybridizationsolution was replaced by a buffer cocktail comprising amplificationprimers, nucleotides, magnesium chloride and the enzymes or enzymaticactivities necessary for amplification. (These enzymatic activities are:reverse transcriptase activity; RNase H activity; and RNA polymeraseactivity. The activities of these enzymes coordinately serve to amplifythe isolated sequences in a fashion similar to that of NASBA.) Once theamplification cycles were completed, the amplified material waselectronically isolated or captured and then quantitated (i.e.,detected) by various methods known in the art. In general, suchdetection may be carried out using, for example, a captureoligonucleotide specific for the newly synthesized region or, afluorescently-labeled oligonucleotide in a “sandwich assay.”

[0175] Each stage of this process is augmented as compared to existingtechnology. For instance, the electronic targeting of the targetsequence followed by its specific hybridization using suitable captureoligos (e.g. the primers for amplification) allows for the electronicremoval of unwanted or contaminating DNA or RNA. The removal ofnonspecific nucleotides that can cause non-specific binding andamplification, allows for a higher complexity of amplification events tosimultaneously occur, as well as for more specific amplification. Inaddition, if all the primers for amplification are anchored,amplification events using different target sequences can occursimultaneously at different locations upon the chip or device, i.e.multiplex reactions. The enzymes themselves can also be targeted,allowing for greater precision in mediating the amplification events orstages. Finally, the products of the amplification reaction can also betargeted to alternative sites and quantified, allowing the progress ofthe amplification reaction to be followed.

[0176] In one representative, but not limiting experiment, NASBAamplification of an HTLVl plasmid was performed in solution using threedifferent concentrations of template plasmid (approximately 1 ng, 1 pg,and 1 fg). The reaction employed an initial melting of the DNA templateat 95° C., followed by an isothermal annealing step of 15 minutes at 50°C. The annealing reaction consisted of 8 μl of 2.5× NASBA mix (100 μL of25 mM NTP mix, Pharmacia Lot #60920250111; 50 μL of 25 mM dNTP mix,Pharmacia Lot #6092035011; 50 μL of 1M Tris, pH 8.5; 31.25 μL of 2M KCl;15 μL of 1M MgCl₂; and 253.75 μL sterile H₂O), 1 μl of a 5 μMconcentration of an oligonucleotide primer (#885; 5′ AATTCTAA TACGACTCACTATAGGGAGA GGTGATCTGA TGTCTGGAC AGG 3′ (SEQ ID NO. 16), and 1 μl of oneof the three dilutions of the HTLV1 plasmid (or none) in four separatetubes to achieve 1 ng, 1 pg, 1 fg, and 0 final concentrations. Enzymeswhich would not survive the 95° C. denaturation step were added at thebeginning of the amplification step. Thus, 1 μL of 100 mM DTT(dithiothreitol) and then 0.5 μL AMVRT (AMV reverse transcriptase fromBoehringer Mannheim (Cat No. 1495062; Lot No. 83724624-76) were added atthe 50° C. step. The reaction was terminated by heating to 95° C. for 5minutes. The tubes were placed on ice.

[0177] Following the annealing reaction, an amplification reaction wasset up, also in four tubes, consisting of 10 μL 2.5× NASBA mix; 1 μL of250 mM DTT; 0.3 μL of Rnase H (Ribonuclease H from Boerhinger Mannheim,Cat No. 786349; Lot No. 13656445-05); 2.5 μL enzyme mix (20u T7polymerase from Boerhinger Mannheim Lot # 83495822-31; 8 u AMV RT; 0.2 uRNase H; and 2.5 μg Rnase and Dnase free BSA (Bovine Serum Albumin) fromPharmacia # 6078914011); 6 μL of primer mix (5 μL of 5 μM primer #885; 5μL of 5 μM primer #882: ACTTCCCAGGGTTTGGACAGAGT (SEQ ID NO. 17); 18.75μL 100% DMSO; and 1.25 μL H₂0); and 2 μL of primed DNA from the fourannealing reaction tubes, each placed in a separate tube. The reactionwas incubated for 60 minutes at 40° C., then put on ice. The reactions(10 μL) were than separated on a 2% agarose gel and stained withethidium bromide.

[0178] The highest concentration of starting template plasmid producedthe largest amount of product, whereas the lower two concentrationsproduced little or no product (FIG. 10a). The product of the 1 ngreaction (the bright band on the gel) was cut out of the gel and thendiluted either 200-fold, 500-fold or 1000-fold in 50 mM histidine. Thereaction product of the 1 pg template reaction was also diluted 200-foldfor comparison. These reaction product dilutions were thenelectronically targeted to capture sites upon a microarray containingeither specific (500 μM of XL5R.bio, 5′ TTCTTTTCGGATACCCAGTCTACGTGTTTG3′ (SEQ ID NO. 18) or non-specific (ATA5.bio) pre-targeted captureantibodies using 500 μA constant current for 1 minute, changing thebuffer and targeting the next capture site without washing. Aftertargeting the reaction products, the capture sites were washed 5× withhistidine (50 mM) and the fluorescence at each location evaluated (FIG.10b) using a fluorescently labeled reporter oligo (HTVPXs.313TR;(NH2)-ACTTCCCAGGGTTTGGACAGAGT 3′ (SEQ ID NO. 19) 15 μL) introduced,passively, for 15 minutes at 25° C. Following 3 washes in 1 mL of 0.2×STE/i % SDS, and 5 additional minutes in STE/SDS, the capture sites wererinsed, and a 2 second image was taken. Thereafter, the buffer waschanged to histidine and the capture sites were run by column at 200μA/pad for 1 minute, washed, and a 2 second image taken. The results ofthe electronic sandwich assay of the amplified reaction paralleled therelative amounts of amplified product introduced, as shown in FIG. 10b.

EXAMPLE 3

[0179] In yet another example, anchored SDA is carried out, preferablyusing electronic targeting of the target nucleic acid to the specificsite, and preferably including at specific sites upon the arraynon-cleavable oligonucleotides in combination with a greater ratio ofnormal SDA primers (i.e. the non-cleavable primers do not contain therequisite restriction endonuclease site necessary for SDA, but which areidentical to SDA primers in other aspects). Anchored SDA is then carriedout, using electronic targeting of template nucleic acid to the specificsite followed by amplification and reporter hybridization. The optimalratio of non-cleavable to normal primers is determined empirically, andis based on the signal obtained from reporter labels. Alternatively,other sites and/or functionalities can be introduced upon thesenon-amplifying primers for the purposes of subsequent cleavage andanalysis or other manipulations. The prime criteria of thesenon-cleavable primers is that the 3′ terminus contains sufficienthomology to the target nucleic acid or amplified products thereof tohybridize and serve as the basis for primer extension by polymerase.

[0180] In a specific experiment of this example, different proportionsof standard Factor V amplifying primers were mixed with primers which nolonger had a BsoBI site present. These mixtures were targeted todifferent locations upon the array and diluted Factor V PCR ampliconswere targeted to each location. The entire array was then washed and amixture containing SDA amplification reaction components (exceptamplifying primers) was added. The amplification reaction was allowed toproceed for 30 minutes at 60° C. then, following denaturation,Bodipy-Texas Red labeled reporter probes were added and hybridized. Thefluorescence present at each site was then quantified.

[0181] The experimental protocol followed in this experiment was asfollows. First, microchips were prepared for electronic addressing andhybridization by scraping any agarose away from the outer electrodes andtreating each microchip surface with Rain-X. The chips were washed threetimes with water and allowed to stand in water for at least about 30minutes. Then Factor V SDA primers (i.e., Seq. I.D. Nos. 20 and 21) andnon-cleavable (NC) primers (i.e., Seq. I.D. Nos. 42 and 43) were dilutedto 2 μM total (from 0-100% non-cleavable primers mixed with SDA primers,see Table III below) and equal volumes of 100 mM histidine were added tomake a 1 μM primer solution in 50 mM histidine buffer. Next 10 nMbtr-T12 and 1 μM ATA-5 oligos were prepared as controls in 50 mMhistidine. Factor V template DNA was then diluted to an appropriateconcentration and incubated at 95.5° C. for about 5 minutes. An equalvolume of 100 mM histidine was added to make a final concentration of 50mM histidine buffer. TABLE III Non-Cleavable Primers to SDA Primers Mix% Non-Cleavable Primers 2 μM NC Primers (μl) 2 μM SDA Primers (μl)  0  0100  10  10  90  20  20  80  30  30  70  40  40  60  50  50  50  60  60 40  70  70  30  80  80  20  90  90  10 100 100  0

[0182] The SDA/Non-Cleavable primers mix, as well as controls, were thenelectronically addressed and a template was hybridized onto eachmicrochip array. An image was taken and the microchips were washed threetimes with water and incubated with 1 mg/ml BSA for 30 minutes at roomtemperature. The microchips were then washed two times with water andpre-incubated at 60° C. for 5 minutes in a humidifying chamber (i.e. apetri dish with moistened Whatman 3 MM paper).

[0183] An SDA mix comprising 40 mM K₂HPO₄, 1.6 mM dCTPαS, 1.6 mM DTTP,1.6 mM dCTP, and 1.6 mM dGTP, 8.3 MM MgCl₂, 1.3 units BsoBI enzyme, and0.5 units Bst polymerase, was pre-incubated at 60° C. for 5 minutes.Water was removed from the microchips and 10 μl of pre-warmed SDA mixwas added to each microchip without allowing the microchips to cooldown. The microchips were then incubated at 60° C. for 30 minutes. TheSDA reaction was then stopped by removing the solution from eachmicrochip and transferring it to an eppendorf tube containing 2 μl 100mM EDTA. The supernatant was then analyzed on non-denaturingpolyacryamide gels.

[0184] The microchips were washed with 0.5× SSC solution, wherein theSSC solution comprises 75 mM NaCl and 7.5 mM NaCitrate, pH 7.2, at leastthree times. Next, the microchips were incubated in 0.5× SSC, pH 12solution for 4 minutes, with the solution being pipetted up and downabout every minute. Each microchip was washed at least three times with0.5× SSC, pH 7.2, then three times again with 4× SSC solution. Passivehybridization of 1 μM reporter oligonucleotides in 4× SSC was thencarried out at room temperature for 3 minutes. Each microchip was washedextensively with 4× SSC. If necessary, an additional stringent wash with0.2× SSC/1% SDS was done for 5 minutes at room temperature. Themicrochips were then washed extensively with 0.2× SSC. Finally, themicrochips were imaged with appropriate lasers and filters, and thefluorescence present at each site was quantified. Results from thisexperiment are shown in FIG. 11. As shown in FIG. 11, a 10% optimalpercentage of non-cleavable SDA primers included in the SDA primer mixfor anchored SDA gave an approximately 2-fold increase in specificsignal over the absence of non-cleavable primers (0%). As expected, withan increase in non-cleavable to SDA primer ratios, the efficiency of theSDA reaction decreases to levels where no detectable SDA amplificationcan be seen. This demonstrates that the addition of non-cleavableprimers to the SDA primer mix, which in effect retains any signal thatmay have been nicked prior to denaturation of the double-strandedtemplate, improves signal intensity in anchored SDA.

EXAMPLE 4

[0185] In another embodiment, an amplification method of the presentinvention comprises an allele-specific SDA method. The method preferablyselectively amplifies only those strands that include a specific allele.The method preferably utilizes amplifying primers designed so that theirrespective 3′ termini include nucleotide bases that are complementary tothe nucleotide sequence of the desired allele. At least one of theprimers may also preferably include a biotin moiety on its 5′ end toprovide a facile mechanism for capturing amplicons on the arrayfollowing electronic targeting and amplification. Generally, thespecificity of the process of this example is derived from the lowefficiency of nucleic acid chain extension when the 3′ terminalnucleotide of the primer is non-complementary to the target sequence.

[0186] In a modification of this example, individual amplified patientnucleic acid samples are immobilized in discrete locations on themicroarray, and all samples are probed simultaneously with gene orallele-specific reporter probes. Individual patient samples areimmobilized by introducing biotin into the samples during SDA. One ofthe SDA primers is added which contains a 5′ biotin linker which doesnot have a restriction cleavage site, and, therefore, is not cleavable.The samples are denatured and addressed to individual capture sites. Asingle stranded amplicon from each patient is immobilized at anindividual capture site. Once all patient samples are immobilized, theyare all probed simultaneously and in parallel. Thus, an open microchipis used to analyze multiple patient samples with minimalcross-contamination.

[0187] In this example, the biotinylated primer is preferably either anoncleavable version of the flanking primer used for amplification, oran internal sequence. In either case, it forms a dead end product (i.e.one which is not further amplified). The primer is preferably present inlimited amounts so that the entire primer is converted to product. Forinstance, when screening for a genetic mutation such as, for example,the Factor V Leiden mutation, there are only two alleles, a wild typeand a mutant. Amplification is performed using primers which arespecific for the wild type locus, but not the allele (i.e., mutant). Theinternal biotinylated primer is converted to a product shorter than thefull length amplicon through extension if the allele is present. Thefragment is then addressed to a pad and subsequently probed with anallele-specific probe, or an allele-specific biotinylated internal probeis used. Amplification may take place in the presence of fluorescentlylabeled nucleotides. Preferably, each patient sample is amplified in twoseparate reactions with allele-specific primers (for wild and mutantalleles) which are then addressed to different pads, or the tworeactions are performed simultaneously using reporter molecules thatfluoresce in two different colors and both products are addressed to thesame capture site (in which case genotype would be determined by thefluorophore remaining at the site for that patient).

[0188] (See Example 1B for additional embodiments of allele-specificmethodology and technique).

EXAMPLE 5

[0189] In another embodiment, SDA products may be simultaneouslygenerated and specifically captured on a microchip by performingthermophilic SDA (tSDA) in a flow cell region over the microchip of thepresent invention. (see U.S. Pat. No. 5,648,211 for a discussion on tSDAand U.S. Pat. No. 5,547,861 for a discussion on signal primer extension,both herein incorporated by reference). By tSDA is meant SDA usingthermophilic enzymes allowing operation at temperatures in excess of 40°C. to facilitate stringent hybridization. Prior to amplification aninternal capture sequence having a 5′ biotin modification is immobilizedpreferably to a specific streptavidin-containing capture site location.As single stranded amplicons are generated free in solution during theSDA process, a fraction of the amplicons specifically hybridize to theimmobilized capture oligonucleotide. Detection of the hybridized strandis preferably via one of the methods described throughout thisdisclosure. This embodiment of the method allows use of very smallsample volumes (e.g., on the order of about 10 μl), and allows forspecificity controls due to use of sequences for capturing that arepreferably located on separate capture sites and are internal to thesequences used to perform SDA priming. Moreover, detection of thecaptured sequences may occur in “real time” as they are being generatedduring the SDA reaction thereby facilitating the simultaneous SDA andmonitoring of the SDA reaction and generated amplicons.

[0190] With respect to this method there are two exemplary schemes toincorporate a fluorescent species for detection. In a first scheme toincorporate a fluorescent species for detection, as is shown in FIG. 5A,an additional oligonucleotide 36 is included in the amplificationreaction. This additional oligonucleotide is fluorescently labeled andbinds to its single stranded complemer generated by the amplificationprocess. Upon binding, polymerization is initiated in a 5′ to 3′direction from this primer by the polymerase 37 used in the SDAreaction. As a course of the regular amplification process, anoligonucleotide which functions as an amplifying primer binds 5′upstream to the same strand as the fluorescently labeled species. Aspolymerase extension occurs from this primer, the fluorescently labeledstrand is displaced and released as a single stranded species free intosolution above the array. On the array are previously addressed anchoredcomplementary oligonucleotides. These serve to capture a portion of thefluorescently labeled oligonucleotides and provide a fluorescent signalupon the array which is both location-specific (and, therefore, sequencespecific) and increasing over the course of the reaction.

[0191] In a second scheme to incorporate a fluorescent species fordetection, as is shown in FIG. 5B, anchored capture oligonucleotideshave either an unmodified endonuclease restriction sequence and capableof supporting an SDA reaction 40 or a modified sequence that will not berecognized by an endonuclease 45. These anchored capture primers 40 and45 are used to bind single stranded products 42 of the amplificationreaction. These capture oligonucleotides 40 and 45 serve as the site foroligonucleotide extension by polymerase activity. Upon completion of theamplification reaction, the double stranded material is melted,preferably by electronic or chemical methods (including, for example,alkaline in pH 12), releasing the original amplicon 42 and extensionproduct 43. The array is washed and then a fluorescently labeledoligonucleotide 44 is introduced. These reporter oligonucleotidesspecifically hybridize only to the polymerase extended portions of thecapture oligonucleotides 40 and 45. In this scheme it is preferred thatthe ratio of cleavable to noncleavable oligonucleotides is about 10:1.It is believed that this ratio allows the amplification reaction tooptimally proceed while providing a sufficient number of uncleavedextension products remaining at the capture sites for detection byreporter probe.

EXAMPLE 6

[0192] In still another embodiment of the invention, SDA is preferablyconducted directly on an electronically addressable microchip under thefollowing conditions. The sample is initially prepared and randomlysheared to less than about 5 kB. The sample is then denatured and targetnucleic acid is captured to a single capture site that contains both 5′and 3′ SDA primers. “Bumper primers” which hybridize to the regionsimmediately upstream of the capture primers are added in a relativeconcentration of about {fraction (1/10)} that of the captureoligonucleotides. An SDA mix (i.e. 3 unmodified dNTPs, 1 thiol modifiedNTP, (and, possibly, a fluorescent labeled NTP,) and enzymes preferablycomprising thermophilic exo (−) DNA polymerase plus restriction enzyme)are passively added. The microchip is then heated to about 40-60° C. andSDA is allowed to proceed.

[0193] “Real time” detection of the SDA reaction and product ampliconsis possible by incorporating NTPs which allow fluorescent energyexchange or quenching. For example, an NTP containing Bodipy Texas redis combined with one that contains Cy5. Incorporation of NTP viapolymerase elongation can be continuously monitored by monitoringfluorescent energy shift.

[0194] Under one theory it is believed that the preferred minimumestimated spacing between adjacent oligonucleotides on a pad is about1.25 nm (10⁴ ODNs/80 μm²=100×10⁶/80×(10³)² nm²=1.25 ODN/nm²). Ifoligonucleotide bridging is required to start SDA, then it is believedthat the optimal length of an SDA fragment which will allow optimalamplification can be determined empirically. As a starting point, 100bp=34 nm seems reasonable.

EXAMPLE 7

[0195] In this embodiment, a novel method of an anchored SDA reactionwhich alters the spatial relationships between amplification primers,target DNA, and enzyme molecules is provided. Because both amplificationprimers are brought into close proximity to one another, the efficiencyof the SDA reaction is actually increased. The spacing relationshipbetween the amplification primers may also be adjustable by alteringlinker elements between the primers thereby enabling precise definitionof the stoichiometry ratios of the primers, the local concentration ofthe primers, site directed template capture, and spatial relationshipsof the primers, so as to set up the SDA mechanism in a coupled-concertedfashion to benefit exponential amplification of target DNA.

[0196] Referring now to FIGS. 12 or 15, SDA target capture primers areattached to specific areas or capture sites 5 on an electronicallyaddressable microchip. The capture primers are attached at each sitesuch that both upstream and downstream primer pairs required for SDAspecific for a target nucleic acid of interest are present together inclose proximity to one another at the capture site. With regard to FIG.12, branched structure 3 is attached to capture site 5 and to the 5′ends of plus and minus strand SDA nucleic acid primers. For each primer,an unmodified restriction site sequence 1 (i.e., the unmodified strandof a hemimodified restriction site) is located 5′ to target specificcapture sequences 2 and 4. With regard to FIG. 15, linear plus and minusstrand nucleic acid SDA primers are attached to capture site 5 at theirrespective 5′ ends. Like the branched primer pairs, the linear SDAprimers comprise unmodified restriction site 6 sequence 5′ to targetspecific capture sequences 7 and 8.

[0197] The microchip may be prepared according to teachings known in theart such as the method disclosed in U.S. Pat. No. 5,605,662 hereinincorporated by reference. In the current example, prior to addition ofSDA primers, the streptavidin-agarose layer was scraped from the outerelectrodes of the microchips. The edges of each microchip werewaterproofed with Rain-X (Unelko Corporation, Scottsdale, Ariz.) and thesurface of the microchip buffed and cleaned with a cotton swabapplicator. The microchips were incubated with milli-Q water for about30 minutes at room temperature before use.

[0198] The microchips were then washed with 50 mM histidine buffer andbiotinylated oligonucleotides (e.g., oligo dT12-btr) having afluorophore in 50 mM histidine buffer were addressed to the capturesites using a standard A/C protocol (800 namps for 25 seconds) to checkthe quality of the streptavidin microchips. The btr fluorophore wasimaged using the appropriate excitation and emission filters for btr.The SDA primers (Seq. I.D. Nos. 20 and 21) were addressed to selectedcapture sites using the same standard A/C protocol.

[0199] As shown in FIG. 13, SDA is carried out at capture sites.Following denaturation of the double stranded target species, singlestranded target molecules (e.g., a plus strand 10+ shown in FIG. 13) arefirst addressed to the capture sites. For electronic hybridization ofthe various templates, double stranded DNA target sequence was firstdenatured at 95° C. and mixed with an equal volume of 100 mM histidinebuffer. The template mixture was then electronically hybridized to thecapture SDA primers using a standard A/C protocol for hybridization (1.6μAmps for 60 seconds). After hybridization of the template mixture, themicrochips were washed twice with water and incubated with 1 mg/ml BSAfor 30 minutes at room temperature to block any non-specific bindingsites. The microchips were washed again with water twice and pre-warmedat 60° C. for 5 minutes. All SDA solutions were also pre-warmed at 60°C. for 5 minutes. After pre-warming, the water was removed and themicrochips were incubated with 10 μl SDA reaction mixture (40 mM K₂HPO₄pH 7.6, 1.6 mM each dCTPαS, dTTP, dATP, and dGTP, 8.3 mM MgCl₂, 1.3units BsoBI and 0.5 units Bst polymerase) for 30 minutes at 60° C. in ahumidifying chamber. The reaction was stopped by removing thesupernatant from the microchip surface to an eppendorf tube with 2 μl of100 mM EDTA.

[0200] As indicated in FIG. 13, strand extension of the target nucleicacid of both plus and minus strands undergo strand displacement to formplus and minus single stranded amplicons (e.g., 12− and 13+). The plusand minus strand amplicons may each be electronically hybridized toadjacent or nearby unused primer pair sets.

[0201] In the instant example, following the SDA reaction, themicrochips were washed three times with 0.5× SSC, pH 7.2. The SDAproducts were then denatured on the microchip in situ with addition of0.5× SSC, pH 12.0 for 4 minutes in which the microchips were washed withfresh buffer every minute. The microchips were then washed with 0.5×SSC, pH 7.2 at least 3 times, with 4× SSC, pH 7.2 about 3 times. Themicrochips were then incubated with a 1 μM mixture of btr-labeledreporter oligonucleotides in 4× SSC for 3 minutes at room temperaturefollowed by extensive washing with 4× SSC at room temperature, thenimaged with the appropriate laser and excitation/emission filters.

[0202] Although for simplicity in showing the efficiency of anchoredSDA, this example carries out detection of SDA products followingamplification, detection may be carried out during amplification usinglabeled target specific probes that are blocked at their respective 3′ends such as by incorporating a 3′ phosphate group rather than a 3′ OHon the terminus of the probe. Such labeled probes may further comprisesingle stranded nucleic acids which may be electronically addressed tothe capture sites allowing detection of increasing signal as target andamplicon species are amplified at the capture pad site without the probeitself taking part in the SDA extension or amplification process.

[0203] In addition to the electronically controlled anchored SDAdescribed above, two additional protocols were followed as controlswherein target nucleic acids were captured by passive hybridizationfollowed by anchored SDA, and where SDA was carried out in solution.First, in the passive hybridization experiments, double stranded targetnucleic acids were first denatured at 95° C. for 5 minutes. The solutionwas then brought to a 4× SSC concentration with a 20× SSC (3M NaCl, 0.3M NaCitrate) stock and 20 μl of the mixture was pipetted onto themicrochip (which had been previously electronically addressed with SDAprimers) and incubated at room temperature overnight. Following thetarget hybridization to the primers, SDA experiments were carried out asdescribed above.

[0204] Second, where SDA was carried out in solution, no microchips wereused. The reason for this is that the purpose of conducting solutionbased SDA was to compare the capacity to amplify target species in amultiplex format in solution versus on a microchip. The solution basedSDA experiments were carried out in eppendorf tubes in a total of 50 μlof SDA mix as described above.

[0205] In a first method of this example three different target nucleicacid species were amplified by SDA using primer pairs that wereaddressed to specific locations on an electronically addressablemicrochip. Ultra pure human placental DNA, Chlamydia genomic templateand deoxynucleoside triphosphates were obtained from Becton Dickenson.Target templates for nucleic acids directed to detect the presence ofgene sequence associated with hemochromatosis and Factor V were obtainedusing SDA bumper primers (Seq. I.D. Nos 22 and 23) and human placentalDNA. PCR reaction conditions for amplifying such templates is well knownto one of ordinary skill in the art of amplification. SDA capture primerpairs, bumpers, and signal probes for each test target species weresynthesized and PAGE-purified by Oligos, Etc. (Oreg.). The restrictionsite encoded into the primer sequences was BsoBI.

[0206] The following is a list of the various SDA primers and signalprobes for each of the target species:

[0207] SDA primer biofacVlOsSDA.213, (SEQ ID NO. 20)

[0208] 5 ′[biot]ACCGCATCGAATGCATGTCCTCGGGTCTCTGGGCTAATAGGA 3′

[0209] SDA primer biofacVaSDA.297, (SEQ ID NO. 21)

[0210] 5′[biot]ACGATTCAGCTCCAGACTTCTCGGGTCAGAATTTCTGAAAGG 3′

[0211] bumper primer facVlOs.179, (SEQ ID NO.22) 5′ ACTACAGTGACGTGGACATC3′

[0212] bumper primer facV10a.-127 (SEQ ID NO. 23) 5′TGTTATCACACTGGTGCTAA 3′

[0213] Signal probe facV10a.276 (SEQ ID NO. 24) 5′ [BTR]CTGTATTCCTCGCCTGTC 3′

[0214] SDA primer chlaAL1.4811, (SEQ ID NO. 25)

[0215] 5′ [biot]CACGTAGTCAATGCATGTCCTCGGGTACAACATCAACACCTG 3′

[0216] SDA primer chlaAR1.4858, (SEQ ID NO. 26)

[0217] 5′ [biot]ACGATTCAGCTCCAGACTTCTCGGGTGAGACTGTTAAAGATA 3′

[0218] bumper primer chlaBLI, (SEQ ID NO. 27) 5′ CAGCAAATAATCCTTGG 3′

[0219] bumper primer chlaBRI, (SEQ ID NO. 28) 5′ CATTGGTTGATGGATTATT 3′

[0220] Signal probe chlaDIL.4826, (SEQ ID NO. 29) 5′ [BTR]GTCGCAGCCAAAATG 3′

[0221] Signal probe chlaCP2.4841, (SEQ ID NO. 30) 5′[BTR]TTCCATCAGAAGCTGT 3′

[0222] SDA primer haemsdas.6679, (SEQ ID NO. 31)

[0223] 5′ [biot] CACGTAGTCAATGCATGTCCTCGGGTATAACCTTGGCTGTAC 3′

[0224] SDA primer haemsdaa.6773, (SEQ ID NO. 32)

[0225] 5′ [biot] ACGATTCAGCTCCAGACTTCTCGGGTGCTCTCATCAGTCACA 3′

[0226] bumper primer haempcrs.6596, (SEQ ID NO. 33) 5′TGAAGGATAAGCAGCCAAT 3′

[0227] bumper primer haempcra.6773, (SEQ ID NO. 34) 5°C.TCCTCTCAACCCCCAATA 3′

[0228] Signal probe haemreps.6712, (SEQ ID NO. 35) 5′[BTR]AGATATACGTGCCAGGTG 3′

[0229] Signal probe haemreps.6733, (SEQ ID NO. 36) 5′[BTR]CTGATCCAGGCCTGGGTG 3′

[0230] As depicted in FIG. 16, biotinylated SDA primers for Factor V(FAC V), Chlamydia (CHL) and Hemochromatosis (HC) were anchored ontostreptavidin-containing microchips and a mixture of Factor V, Chlamydiaand Hemochromatosis templates were hybridized onto the primerselectronically. Control template T12 was also anchored.

[0231] Anchored SDA was performed on microchips in situ at 60° C. for 30minutes as described previously and processed accordingly. As can beseen, no SDA amplicons can be detected when template is not hybridizedto the SDA primers on the microchip (FIG. 17). However, when a mixtureof the templates are hybridized to the SDA primers, simultaneousamplification of the three amplicon systems can be seen (FIGS. 18-20).Accordingly, when only one species of template is hybridized in thepresence of all three types of SDA primer, only the area where thecorresponding SDA primer is anchored shows a signal indicatingamplification has taken place. This confirms the specificity, as well asthe flexibility, of the anchored SDA system when done in situ onmicrochips. Interestingly, as shown in FIG. 21, when solution-based SDAis performed using the same three SDA primer sets, multiplexamplification is greatly compromised. Solution SDA was performed onFactor V, Chlamydia and Hemochromatosis separately, as well as togetherin one reaction (ALL) followed by analysis on a 6.0% non-denaturingpolyacrylamide gel. As can be seen, all three systems amplify when doneseparately. However, when all three primer sets and templates arecombined into one reaction, Factor V amplification is greatly depressed.Additionally, when the templates were hybridized to the primers bypassive hybridization, the amplification efficiency was significantlyreduced, possibly due to the inefficient hybridization caused bytemplate reannealing. These results underscore the need in the art for asystem such as that of the current invention for a multiplexamplification system that can perform multiplex amplification anddetection of target species without hindrance as may be observed insolution based and/or passive hybridization systems.

[0232] In a second embodiment of this example, the preparation ofbranched SDA target capture primer pairs may be synthesized by numerousmeans. In a preferred embodiment, the branched moiety may be produced asdescribed below. First, as depicted in FIG. 22, the starting substratefor Y-primer synthesis is a biotin-conjugated lysine with atert-butyloxy carbonyl-protected α-amino terminal. The tert-butyloxycarbonyl (TBC) moiety on the a-amino terminal allows selectiveattachment of the SDA primer arms separately. In this case, the a-aminoterminal is protected but the α-amino terminal can react with carboxylicacid, allowing the SDA sense primer to be attached to the a-aminoterminal end. The α-amino terminal end can then be deprotected withtri-fluoroacetic acid/dichloromethane (TFA/DCM), which removes thetert-butyloxy carbonyl moiety and allows attachment of the SDA antisenseprimer via the carboxylic acid terminal. This attachment sequence allowsthe formation of a Y-primer where both SDA primers are addressed to thebranched moiety at their respective 5′ ends. The resulting Y-shapedprimer pair can then be attached to the streptavidin permeation layer onthe microchip.

[0233] The synthesis of Y-shaped primer pairs for anchored SDA isintended to increase the overall efficiency of the SDA reactiontwofold: 1) by placing the SDA primers in relatively close proximity ofeach other, thereby increasing the rate of interaction between extendedamplicons of one strand and subsequent binding of the cleaved ampliconto the opposite strand primer; and 2) by increasing the density ofprimers in a given area over conventional oligonucleotide SDA primers.In the synthesis protocol above, the Y-primer is attached to themicrochip permeation layer via a streptavidin-biotin bond; however,other amide-bond attachment chemistries can be used, including but notlimited to prolinx, R-SH, or any other functional group onto themacromolecule. The branched primer pairs may be used in carrying out SDAreactions as described above.

EXAMPLE 8

[0234] Still another example provides an asymmetric amplification methodto address the problem of hybridization between sense and antisenseamplicons that are generated during SDA. When using SDA, generally, bothsense and anti-sense strands are generated in equal amounts. Undertypical conditions of amplification, the complementary strands hybridizetogether. However, hybridization of oligonucleotides to specific siteson a microelectronic array (both for hybridization of amplimers tocapture oligonucleotides and detection of hybridized material byfluorescently labeled reporter oligonucleotides) requires generation ofsingle stranded species from the amplicons. Therefore, the complementarystrands that are hybridized together must be separated prior tohybridization to captures upon the array and/or prior to detection bylabeled reporter unless one strand is amplified more than the other(i.e. unless amplification is asymmetric). This is conventionally doneusing heat or chemical denaturation before or after electronicaddressing. Asymmetric amplification removes the need for suchthermocycling step.

[0235] A key feature of asymmetric amplification is the generation of apreponderance of one amplicon over its complementary amplicon sequence.In a solution environment, this method is typically accomplished byhaving a disproportionate ratio of amplifying primers. In the initialstages of the amplification process, the effective concentration of thesense and antisense amplifying primers being in large excess to templateproduces an environment conducive to exponential amplification of theoriginal double stranded template material. As the reaction proceeds,the amplifying primer originally present in lesser amounts iseffectively exhausted thereby leading to conditions of linearamplification by the primer remaining in excess. The particular effectof the polymerase mediated displacement of amplified material during SDAensures that this linearly amplified material is free in solution andavailable for hybridization without the necessity for denaturation ofdouble stranded species. With respect to objects of the invention, analternative approach is to place both primers in solution at the sameconcentration, but to add a competitor that partially inhibits, or“poisons” generation of one strand. Over time this will also lead to apreponderance of one strand of the amplified target.

[0236] Where capture probes are anchored, creation of predominantly onestrand can be enhanced by designing anchored capture probes that arecomplementary to one strand of the amplicons being generated andreleased free into solution. In a preferred embodiment, the captureprobes are different from normal SDA primers in two respects. First,they preferably do not possess a functional restriction site, therebyblocking the endonuclease nicking/polymerase extension-displacementsteps. Second, the 3′ ends of the capture probes preferably are notsuitable for extension by polymerase activity. During SDA this modifiedcapture primers will hybridize to amplicon strands effectively pullingthem out of the SDA pathway so that they will not be available forfurther amplification. The capture of such single strands may bedirected to occur at a capture site located at a remote position fromthe site where SDA is occurring. Thus, a bias in the strandedness of theamplicon population will be generated, which is an effective form ofasymmetric amplification due to limiting the quantities of one strand ofamplification product.

[0237] In another example asymmetric amplification may be enhanced byincluding in the SDA reaction a competitive inhibitor of one of theprimers of a given set of primers. As in the above example, thecompetitive primer is preferably either non-extendible or non-cleavable.The inclusion of the competitive primer biases the reaction toward thecreation of single-strands through a linear reaction process.

[0238] Oligonucleotide sequences are rendered non-extendible usingvarious means including blocking the 3′ OH end, and mismatching the 3′terminal nucleotides(s) with respect to the template sequence.Oligonucleotide sequences are rendered non-cleavable by modifying theoligonucleotide backbone through the inclusion of modified linkages suchas phosphorothioates or more simply by changing the sequence at therestriction endonuclease recognition site. Probes modified as suchremain fully competent for hybridization. The sequence of the competitoris preferably identical to (or nearly identical to) that of one of theamplification primers. The competitor can therefore compete with theamplification primer for hybridization with a target sequence. Whenbound to the target sequence, the competitor either (1) cannot beextended by DNA polymerase, or (2) can be extended to produce a copy ofthe target sequence. In the case where the competitor is extended, thecompetitor is modified such that resultant copies of the target sequencecannot be cleaved by a restriction enzyme. Different types ofcompetitors are used depending on the amplification method being used.

[0239] In PCR, the competitor is modified such that it cannot beextended. Appropriate modifications are described above. In each cycleof the reaction, the competitor will compete with one of the PCR primersfor hybridization to available target sequences. For example, in areaction where the competitor is added at 10% the concentration of thePCR primer, roughly 10% of hybridization events will be abortive in thatan extension product cannot be produced. The opposite PCR primer is freeto hybridize to all available target sequences and be extended.Therefore, a bias of about 10% in the relative number of the twoextension products is produced in any given cycle. While a 10% bias inearly cycles may not be significant since target concentration is low,such a bias will produce a high concentration of single-strandedmaterial in late stage cycles (where nM quantities or greater of theextension products are being produced).

[0240] In SDA, several methods are preferable. Use of a non-extendiblecompetitor will bias the production of double-stranded templates whichwill allow the nicking and extension reaction to preferentially produceone of the single-stranded displaced products. Use of an extendible,non-cleavable competitor leads to asymmetry by creating double-strandedproducts that cannot participate in the nicking/displacement reaction.Use of both types of competitors may be optimal as extension productsproduced from the non-cleavable primer become part of double-strandedmolecules when only one strand can be nicked and displaced. (see FIG.14).

EXAMPLE 9

[0241] In this example of the invention, SDA is carried out inconjunction with an electronically addressable microchip wherein theatmospheric pressure of the SDA reaction is elevated.

[0242] Where genomic nucleic acid is used, it is preferred that it becleaved into fragments of between approximately 250-500 bp. This may bedone by a restriction enzyme such as HhaI, FokI or DpnI. The selectionof the enzyme and the length of the sequence should be such that thetarget sequence sought will be contained in its entirety within thegenerated fragments or that at least a sufficient portion of the targetsequence will be present in the fragment to provide sufficient bindingof SDA amplification primers. Other methods for generating fragmentsinclude PCR and sonication.

[0243] The primers used in this method generally have a length of 25-100nucleotides. Primers of approximately 40 nucleotides are preferred. Theprimer nucleic acid sequence should be substantially homologous to thetarget sequence such that under high stringency conditions hybridizationbetween primer and template nucleic acid will occur.

[0244] Target nucleic acid fragments are denatured to render them singlestranded so as to permit binding of the primers to the target strands.Raising the temperature of the reaction to approximately 95° C. is apreferred method for denaturing the nucleic acids. Other methods includeraising pH; however, this will require lowering the pH in order to allowthe primers to bind to the target. Following the formation of singlestranded target molecules, SDA is performed as discussed in the numerousexamples discussed herein. Typically, the SDA reaction includes the useof at least one substituted nucleotide during primer extension tofacilitate nicking of one strand during amplification. The nuclease maybe any nuclease typically useful for SDA as discussed earlier.

[0245] In a preferred embodiment of this method, atmospheric pressure iselevated either before or after all the SDA reaction components arecombined. The pressure is elevated to reduce star activity toeffectively enhance the specificity of the restriction endonuclease forits target. The application of elevated pressure may also increase thespecificity of primer interaction with the template nucleic acid and theoverall rate of reaction of the enzymes employed, thereby reducing thetime required for the SDA reaction while increasing its specificity. Byreducing star activity, template independent amplification is decreasedthereby reducing the competitive consumption of reagents by non-specificamplification.

[0246] Elevated pressure can be supplied during the amplification byvarious methods. For example, the reactions could be run in highpressure vessels. The reactions may also be run by placing the containerin a reaction chamber attached to or part of a high-pressure apparatus(High Pressure Equipment Co., Erie, Pa.). It may be advantageous tooverlay the aqueous reaction media with an immiscible phase, such assilicon oil (Sigma) by which pressure can be applied to the aqueoussolution containing the target nucleic acid, nucleosidetriphosphates,and enzymes. Preferably, the pressure is elevated in the range of about100 to about 500 atmospheres.

[0247] Polymerases useful in this method include those that willinitiate 5′-3′ polymerization at a nick site. The polymerase should alsodisplace the polymerized strand downstream from the nick, and,importantly, should also lack any 5′→3′ exonuclease activity and be heatstable. Polymerases, such as the large fragment of DNA polymerase I andthe exonuclease deficient Klenow fragment of DNA polymerase I and asimilar fragment from the Bst polymerase (New England Biochemicals,Beverly, Mass.) are useful. SEQUENASE 1.0 and SEQUENASE 2.0 (U.S.Biochemical), T5 DNA polymerase, and Phi29 DNA polymerases are alsouseful. Generally, thermophilic DNA polymerases are preferred. Theexonuclease deficient thermophilic Klenow fragment of Bst DNA polymerasefrom Bacillus stearothermophillus (New England Biochemicals, Beverly,Mass.) is most preferred.

[0248] In this method, a single reaction temperature may be employedafter denaturation has occurred, and such temperature should be highenough to set a level of stringency that minimizes non-specific bindingbut low enough to allow specific hybridization to the target strand. Inaddition, use of temperature preferably from about 45° C. to about 60°C. should support efficient enzyme activity. Denaturation of the enzymesand nucleic acid is to be avoided.

[0249] During the SDA reaction cycles, theoretically about 20repetitions or cycles will result in about a 10⁶-fold amplification(i.e., SDA X2²⁰=10⁶). Typically, 10⁸-fold or greater amplification isseen in about 30 minutes of amplification.

[0250] High pressure SDA is beneficial for various uses includinggeneration of high fidelity single-stranded nucleic acid probes orsingle-stranded templates for sequencing. Toward this goal, highpressure SDA can be conducted either with a single primer or using twoprimers wherein one primer is in excess over the other. The result isexcess production of one displaced single strand over the other.

[0251] The presence of the amplified target then can be detected by anynumber of methods. One method is to detect reaction products of aspecific size by means of gel electrophoresis. This method isparticularly useful when the nucleotides used are labeled with aradio-label, such as ³²P. Other methods include labeling the nucleotideswith a physical label, such as biotin. Biotin-containing reactionproducts can then be identified by means of avidin bound to a signalgenerating enzyme, such as peroxidase. Another method is elongation of afluorescently labeled internal primer.

[0252] Detection systems useful in the practice of this inventioncomprise homogeneous systems, which do not require separation, andheterogeneous systems. In each system, one or more detectable markersare used and the reaction or emission from the detection system ismonitored, preferably by automated means. Examples of homogeneoussystems include fluorescence polarization, enzyme mediated immunoassays,fluorescence energy transfer, hybridization protection (e.g., acridiniumluminescence) and cloned enzyme donor immunoassays. Examples ofheterogeneous systems include enzyme labels (such as peroxidase,alkaline phosphatase and beta-galactosidase), fluorescent labels (suchas enzymatic labels and direct fluorescence labels (e.g., fluoresceinand rhodamine)), chemiluminescence and bioluminescence. Liposomes orother sac like particles also can be filled with dyes and otherdetectable markers and used in such detection systems. In these systems,the detectable markers can be conjugated directly or indirectly to acapture moiety or the amplified products can be generated in thepresence of a receptor which can be recognized by a ligand for thereceptor.

[0253] Protocol for Strand-displacement Amplification (SDA) UnderElevated Pressure

[0254] Amplification reactions utilize approximately 100 ng of genomicDNA (Factor V) in a total volume of 50 μl. The genomic DNA (humanplacental DNA; Becton-Dickinson) is denatured at 95° C. for 5 minutesfollowed by centrifugation to collect condensate. Next, 1 μl of SDAprimer mix is added (50 μM each reaction) and incubated at 60° C. for 5minutes. SDA mix (40 mM k₂HPO₄ pH 7.6, 1.4 mM each dCTPαS, dTTP, dATPand dGTP, 8.3 mM MgCl₂, 40 units/rxn BsoBI (New England Biochemicals),15.6 units/rxn Bst polymerase (New England Biochemicals), and 0.05 μMeach SDA bumper primers are added and pre-warmed for 5 minutes at 60° C.followed by addition of the mix to SDA primers and target sample.Silicon oil is added to the top of the reaction tubes and placed in higha pressure chamber. The pressure is elevated to between 100 and 500atmospheres and incubate at 60° C. for 30 minutes. Following thereaction period, the pressure is reduced to atmospheric pressure andstopped by addition of 10 μl of 100 mM EDTA. SDA products are visualizedby electrophoresing on 6% non-denaturing polyacrylamide gels. The gelsare stained with ethidium bromide and photographed underUV-fluorescence.

[0255] Alternatively, it is possible to use a device wherein thetemperature and/or pressure is elevated prior to the addition of thepolymerases and/or restriction endonuclease.

[0256] The use of elevated pressure can also be used in the performanceof anchored SDA, or any SDA procedure as described above. Specifically,when anchored SDA is performed on electronically addressable microchips,elevated pressure should decrease star activity and increase efficiencyby reducing primer independent amplification.

EXAMPLE 10

[0257] In another example, SDA may be used in conjunction withelectronically addressable microchips wherein the SDA reaction is“ligation-dependent” or “ligation-based”. This method involves the SDAamplification of a ligated probe using a pair of universal amplificationprimers. The amplification primers are universal in the sense that theyare designed to amplify all ligated probes in a test reaction whetherthe reaction is multiplexed or directed to a singular target. Theligated probe is formed by ligating together a pair of ligation probesthat have hybridized to a target sequence. No bumper primers arenecessary.

[0258] In another embodiment, a method of ligation-based SDA is providedwhere the method is unassisted by an electronic microchip. In thisembodiment it is not necessary to, inter alia, anchor any primers, whichis a procedure that assists in separating primer sets during multiplexamplification, because the primers are universal—there is no need todirect target sequences to specific primers.

[0259] The following functional descriptions of the oligonucleotidereagents are not intended to define or limit their actual physicalcomposition. Rather, the description merely demonstrates that eachreagent exhibits certain functional characteristics. Thus, it should benoted that the functional regions of a given oligonucleotide reagent mayoverlap, or in fact be co-extensive, as where a specific nucleic acidsequence is able to accomplish more than one function. Additionally, theindividual base sequence in any given oligomer depends upon the targetnucleic acid of interest, the restriction enzyme chosen for use in SDA,or an arbitrary sequence chosen for portions of the amplificationprimers and ligation probes so that a degree of universality can beincorporated into the amplification protocol.

[0260] In operation, as illustrated in FIGS. 23(a-c), the ligation-basedSDA method uses a pair of ligation probes that anneal to adjacentnucleic acid sequences on a target. Functionally, the pair of ligationprobes bind to a target nucleic acid sequence such that they can beligated together while they are annealed to the target to form a ligatedprobe template. Ligation will occur only following hybridization of bothligation probes of a ligation probe pair to a target sequence.

[0261] The first ligation probe can be divided into three functionalregions: a 5′ region able to hybridize to target nucleic acid; a middleregion; and a 3′ region comprising a nucleic acid sequence that is ableto hybridize to the first amplification primer. The second ligationprobe can also be divided into three functional regions: a 5′ regionhaving a nucleic acid sequence identical to nucleic acid sequences foundin the second amplification primer and having a restriction endonucleaserecognition site; a middle region; and, a 3′ region able to hybridize totarget nucleic acid.

[0262] With respect to the amplification primers, the firstamplification primer can be divided into two functional regions: a 5′region containing a restriction endonuclease recognition site and a 3′region that is able to hybridize to the first ligation probe. The secondamplification primer can also be divided into two functional regions: a5′ region that contains a recognition site for a DNA restrictionendonuclease and a 3′ region comprising nucleic acid sequence having thesame sequence as the 5′ region of the second ligation probe.

[0263] The ligation-based SDA reaction comprises a number of componentsteps. In Step 1, the pair of ligation probes anneal to adjacentsequences of single-stranded target nucleic acid such that the secondligation probe hybridizes to the target strand at a position on thetarget that is 3′ to the hybridization position of the first ligationprobe. In Step 2, DNA ligase catalyzes the ligation of the two ligationprobes to form the ligated probe template. In a preferred embodiment,the 3′ end of the ligated probe template is modified to prevent primerextension from that end (FIGS. 23(a-c)).

[0264] In Step 3, the first amplification primer binds to the 3′ end ofthe ligated probe template such that the amplification primers extendbeyond the end of the template forming a 5′ overhang. In a preferredembodiment, DNA polymerase catalyzes new DNA synthesis from the 3′ endof the first amplification primer causing the ligated probe to bedisplaced from the target nucleic acid. This results in the release ofsingle-stranded target nucleic acid and the creation of double-strandedDNA oligonucleotide having a 5′ overhang (labeled Product I, FIG. 23).The release of single-stranded target nucleic acid and the creation ofthe double-stranded oligonucleotide occurs without the assistance ofbumper primers. Moreover, the target single strand becomes available forfurther binding of unligated first and second ligation probes.

[0265] Product I thus comprises a first strand having a sequence from 5′to 3′ corresponding to the ligated probe template, and a second strandcomplementary to the ligated probe template strand with an additionalnucleic acid sequence at its 5′ end corresponding to the 5′ end of thefirst amplification primer. This double stranded DNA molecule is capableof undergoing a series of SDA reactions that produce single stranded DNAmolecules able to be bound and amplified by the universal amplificationprimers. The double stranded DNA molecules that result from thesereactions are also susceptible to amplification. Nicking by arestriction endonuclease, followed by primer extension and stranddisplacement, substantially regenerates the double stranded DNA startingmaterial. Together, these ligation-dependent SDA reactions ultimatelyamplify oligonucleotide sequences corresponding to the ligated probe,thereby allowing the detection of the target sequence. These reactionsare described in detail below.

[0266] In Step 5, Product I is nicked by a restriction enzyme to createProduct II. In Step 6, Product II undergoes primer extension and stranddisplacement from the nick, resulting in Product III and Product IV.Product III is essentially the same as Product I except that the firststrand of Product III (which corresponds to the first strand of ProductI) contains an additional sequence at its 3′ end complementary to the 5′end of the first amplification primer. Product IV is a single-strandedmolecule with a sequence comprising the first strand of Product IIlocated 3′ to where this strand was nicked by the restrictionendonuclease.

[0267] In Step 7, Product III is nicked by a restriction endonuclease tocreate Product V. In Step 8, Product V undergoes primer extension andstrand displacement to create Product VI and Product VII. Product VI isessentially the same as Product III. Product VII is a single strandedDNA molecule comprising the nicked strand of Product V located 3′ to thenick site.

[0268] In Step 9, the second amplification primer binds to Product VII.In Step 10, Product VII undergoes a primer extension reaction in bothdirections to create Product VIII. Product VIII is a double strandednucleic acid molecule, the first strand having a sequence correspondingto product VII plus an additional 3′ sequence that is complementary tothe 5′ region of the second amplification primer, and a second strandthat is complementary to the first strand. In Step 11, Product VIII isnicked with a restriction endonuclease to create Product IX. Product IXis essentially the same as Product VIII except that the 5′ end ofProduct IX contains a nick in the nucleic acid corresponding to the 5′region of the second amplification primer. In Step 12, Product IXundergoes primer extension and strand displacement to create Products Xand XI. Product X is the same as Product VIII. Product XI is a singlestranded nucleic acid molecule with a sequence corresponding to thesequence 3′ of the nick, on the nicked strand of Product IX. In Step 13,Product XI is bound by the first amplification primer and in step 14,primer extension in both directions results in Product XII. Product XIIis a double stranded nucleic acid molecule similar to Product III in thesense that it can enter the above described reaction pathway followingstep 6 and prior to step 7. Thus, an initial reaction product of theligation-dependent SDA pathway is ultimately substantially regenerated.

[0269] As described earlier, the SDA reaction may be carried out usinganchored probes. With regard to ligation-based SDA, the anchored probesare preferably either one or both of the amplification primers or one orboth of the ligation probes.

[0270] Experimental data for Example 10

[0271] Experiment 1

[0272] In this example, a general protocol for the preferredligation-based SDA of a target nucleic acid is provided. Concentrationsand volumes of reaction components, and time and temperature profilesmay be adjusted as necessary. Volumes assume a 25 μl ligation reactionvolume and a 50 μl final reaction volume for SDA.

[0273] In a 250 μl microcentrifuge tube, a 5 μl aliquot of an aqueousligation probe solution is added such that the final concentration ofeach probe in a 25 μl lligation reaction volume will be 5 nM. Next, 10μl of a solution of non-specific (carrier) DNA (e.g., Calf thymus DNA isadded to a final concentration of 20-100 μl/ml. Next, 5 μl of the samplecontaining the template nucleic acid (e.g. Cell lysate or purifiedgenomic DNA) at an appropriate concentration is added and the tube isplaced at 60° C. for 3 minutes to allow temperature equilibration.Following equilibration, 5 μl of a solution containing a thermostableDNA ligase is added along with sufficient 5× strength mixture of buffercomponents necessary to allow function of the DNA ligase, and to allowprobe hybridization. See Table IV.

[0274] The 25 μl ligation reaction is incubated at 60° C. for 15 minutesand then 20 μl of an SDA stock mix containing additional buffercomponents, dNTPs, and amplification primers, is added to give finalreaction concentrations (in 50 μl) as shown in Table V. In oneembodiment an additional step is included where the reaction is heatedto 95° C. for 3 minutes to denature the ligated probes from the templateand then the tube is equilibrated at 60° C. for 3 minutes. To thisreaction mixture 5 μl of liquid containing the SDA enzymes is added togive the following final concentrations in a 50 μl final reactionvolume:

[0275] BSOB1 restriction enzyme: 0.8 enzyme units/μl (40U/rxn)

[0276] Bst DNA polymerase: 0.32 enzyme units/μl (16 U/rxn)

[0277] The reaction mixture is incubated at 60° C. for 30 minutes thenthe reaction is stopped by placing the reaction mixture on ice. TABLE IVFinal Concentration in Reaction for Each Ligase Taq DNA ligase Pfu DNAligase Buffer Component (1 U/rxn) (0.2 U/rxn) Tris-HCl pH 7.6 10 mM 10mM Potassium Acetate 25 mM 25 mM Magnesium Acetate 10 mM 10 mMDithiothreitol 1 mM 1 mM Nicotinamide adenine dinucleotide 1 mM NONEAdenosine triphosphate NONE 10 μM

[0278] TABLE V Final Concentrations in 50 μL Reaction (Note: includescontribution SDA Component from ligation reaction) Potassium phosphate35 mM Bovine serum albumin 80 μg/ml Magnesium acetate 10 mMDeoxynucleotide triphosphates (equal 1.4 mM mixture of dATP, dQSTP,dGTP, TTP) Amplification primers (S1 and S2) 250 nM

[0279] Experiment 2

[0280] In this further example, the Salmonella spaQ gene (a portion ofwhich is indicated on FIG. 23d and designated SEQ. ID. No. 41)potentially present in a sample is amplified. The reaction protocol asdescribed in Experiment I is followed using the ligation probes LPI(SEQ. ID. No. 37) and LP2 (SEQ. ID. No. 38) and amplification primers Si(SEQ. ID. No. 39) and S2 (SEQ. ID. No. 40) which are illustrated in FIG.23(d). The example described in Experiment 2 is intended to have generalapplicability. One could create different target-specific ligationprobes for use with the amplification primers S1 and S2 by replacing thesequences of ligation probes L1 and L2 complementary to the spaQ genewith sequences complementary to another target nucleic acid of interest.Moreover, amplification primers S1 and S2, such as those depicted inFIG. 23(d) may be used in a multiplex amplification of more than onetarget nucleic acid.

[0281] Experiment 3

[0282] At high concentrations of ligation probe, ligase may catalyze theligation of the ligation probes in a target-independent manner. Theresulting ligated probe can support SDA and may thus create a falsepositive signal. In this further example, a preferred aspect ofligation-dependent SDA is described where this problem is overcome byrendering the ligation probes initially incapable of being ligatedtogether by ligase. In this embodiment, a pair of unligateable probes isrendered ligateable to allow target-specific, ligation-dependent SDA.

[0283] Generally, the amplification of a background molecule that istarget independent may be prevented by modifying the ends of theligation probes that are involved within the ligation junction. This cantake place in several ways. One such modification involves themodification (including removal, blocking, etc.) of the 3′ hydroxylgroup present on the 3′ terminal nucleotide of the second ligation probe(the upstream probe). Another such modification involves themodification (including removal, blocking, etc.) of the 5′ phosphategroup present on the 5′ terminal nucleotide of the first ligation probe(the downstream probe). Various methods have been and can be devisedwherein the removal and or alteration of these modifications occurspreferentially in the presence of target DNA.

[0284] Specifically, one aspect of this example provides for modifyingthe 3′ hydroxyl group present on the 3′ terminal nucleotide of thesecond ligation probe (the upstream probe) to prevent blunt end ligationbetween the ligation probes. The modified unligateable probe is renderedligation competent using an endonuclease, preferably Endonuclease IV.This reagent is able to excise 3′ terminal nucleotides fromoligonucleotides and thus is used to excise the 3′ terminal nucleotideof the second ligation probe to reveal a new 3′ terminal nucleotide witha 3′ hydroxyl group. This reaction is more preferred when the ligationprobe substrate is associated with target DNA and less preferred whenthe ligation probe substrate is unassociated with other DNA molecules.Consequently, once the ligation probes are bound to target DNA, theendonuclease (preferably Endonuclease IV) is able to excise the 3′terminal nucleotide of the second ligation probe to reveal a new 3′terminal nucleotide with a 3′ hydroxyl group. The free 3′ hydroxyl groupof the second ligation probe, along with the free 5′ phosphate group ofthe first ligation probe, are now substrates for ligation by DNA ligase.

[0285] Since endonuclease tends to operate more efficiently when thesubstrate oligonucleotide is double stranded it will preferentiallyexcise the 3′ terminal nucleotide of the second ligation probe when thisprobe is bound to target DNA, not when it is free in solution. Becausethe endonuclease preferentially renders the initiallyligation-incompetent ligation probes ligation-competent when they are inthe presence of target DNA, the target independent amplification ofbackground molecules is decreased.

[0286] Another aspect of this example provides for the modification(including removal, blocking, etc.) of the 5′ phosphate group present onthe 5′ terminal nucleotide of the first ligation probe (the downstreamprobe) to prevent blunt end ligation between the ligation probes. Themodified unligateable probe is rendered ligation-competent using a DNApolymerase with exonuclease activity. This reagent will allow DNApolymerization (new DNA synthesis) to occur from the 3′ end of theupstream probe (the second ligation probe) into the 5′ end of thedownstream (first ligation) probe. When the polymerase contacts the 5′end of the first ligation probe it will begin to excise nucleotides fromthe 5′ end. As it excises nucleotides from the first ligation probe,nucleotides are added to the 3′ end of the second ligation probe. Inessence, this moves the “gap” between the first and second ligationprobes, the junction to be ligated by ligase, from 5′ to 3′. Bycontrolling the amount and/or type of free nucleotide present insolution, the degree of excision and replacement can be limited.Following dissociation of the polymerase the junction contains a free 3′hydroxyl group and a free 5′ phosphate group, both of which aresubstrates for ligation by DNA ligase. As indicated above, this reactionis more preferred when the ligation probe substrate is associated withtarget DNA and less preferred when the ligation probe substrate isunassociated with other DNA molecules. Again, this is because thereaction that renders the ligation probes ligateable prefers that theligation probes be annealed forming dsDNA. As is understandable to oneskilled in the art, such annealing is preferred for target DNA ratherthan annealing to non-target DNA. Thus, independent amplification ofbackground molecules is decreased.

[0287] Yet another aspect of this example provides for blocking ligationusing base-paring mismatching. Here, ligation is prevented between thefirst and second ligation probes by having the 5′ end of the downstream(first) probe contain one or more mismatched bases. If a probe is saidto contain a mismatched base, it should be understood to mean that theprobe contains a nucleotide that is not complementary to target DNAsequences, in a region of the probe otherwise complementary to thetarget DNA. Mismatched bases prevent ligation by DNA ligase until themismatched bases are excised, as in the above stated example, with DNApolymerase.

[0288] To demonstrate the exonuclease/ligase-dependent SDA (XL-SDA)aspect of this invention, as described in this further example, the ninesets of ligation probes shown in Table VI were synthesized. These probeswere designed to identify the various bacterial species shown. Theprobes have regions complementary to the specific bacterial genes andregions designed for SDA amplification primer binding. TABLE VIBacterial Genus/Species/ gene, Serotype Ligation probe 1 Ligation probe2 product identified (5′-3′) (5′-3′) stx₁, Shiga toxin- GAGGGCGGTTTAATAA(SEQ. ID. No. 45) CGATTCCGCTCCAGACTT (SEQ. ID. No. 46) Shiga-liketoxin-I producing E. coli TCTACGGTGGTCGAGT CTCGGGTGTACTGAGATC (STEC) andACGCCTTAA CCCTTGTCAGAGGGATAG Shigelia ATCCAGAGG dysenteriae type I stx₂,STEC GATGGAGTTCAGTGGT (SEQ. ID. No. 47) CGATTCCGCTCCAGACTT (SEQ. ID. No.48) Shiga-like AATACAATGTGGTCGA CTCGGGTGTACTGAGATC toxin-Il GTACGCCTTAACCCTGGTTTCATCATATCT GGCGTT eaeA, E. coli O157:H7 GACGCTGCTCACTAGA (SEQ.ID. No. 49) CGATTCCGCTCCAGACTT (SEQ. ID. No. 50) intiminTGTCTAGGTCGAGTAC CTCGGGTGTACTGAGATC GCCTTAA CCCTGGTTATAAGTGCTTGATACTCCAG spaQ, Salmonella GATGATGTCATGTTGC (SEQ. ID. No. 51)CGATTCCGCTCCAGACTT (SEQ. ID. No. 52) surface species AATGTCCTGGTCGAGTCTCGGGTGTACTGAGATC antigen- ACGCCTTAA CCCTCATTTAACTATCCC presentingGTCTCGT protein gnd, 6- Salmonella typhi, GAGTAATTACCGTCTT (SEQ. ID. No.53) CGATTCCGCTCCAGACTT (SEQ. ID. No. 54) phospo- SalmonellaCATCTTTTTTTGGTCGA CTCGGGTGTACTGAGATC gluconate paratyphi GTACGCCTTAACCCTGGCTTCATCAAGAA dehydro- TAACATCTATC genase ipaH, Shigella speciesGATTTACGGACTGGTT (SEQ. ID. No. 55) CGATTCCGCTCCAGACTT (SEQ. ID. No. 56)patho- and CTCCCTTGGTCGAGTA CTCGGGTGTACTGAGATC genicity- enteroinvasiveE. CGCCTTAA CCCTTCAGAAGCCGTGAA associated coli GAGAATG gene sodB,Campylobacter GACCAAAACCATCCTG (SEQ. ID. No. 57) CGATTCCGCTCCAGACTT(SEQ. ID. No. 58) superoxide species AACCATGGTCGAGTAC CTCGGGTGTACTGAGATCdismutase GCCTTAA CCCTTTCTAGTTTTTGATT TTTAGTATTATA asd, Vibrio speciesGAGTAGAGGTATGTGA (SEQ. ID. No. 59) CGATTCCGCTCCAGACTT (SEQ. ID. No. 60)aspartate TGAGCCAATGGTCGAG CTCGGGTGTACTGAGATC semi- TACGCCTTAACCCTCTTTGGCTAAACTC aldehyde GGTTTTC dehydro- genase lcrV, Yersiniaspecies GATTAGCTGAGCTTAC (SEQ. ID. No. 61) CGATTCCGCTCCAGACTT (SEQ. ID.No. 62) Yersinia CGCCGTGGTCGAGTAC CTCGGGTGTACTGAGATC V-antigen GCCTTAACCCTCCGTAGCAAGTTGC GTGAAG

[0289] The probes were added to identical sets of ligation-SDA reactionsuch that the number of ligation probe sets in the reactions increasedin the order: I set (spaQ), 5 sets (spaQ, stx₁, stx₂, sodB, ipah), 6sets (as 5+lcrV), 7 sets (as 6+asd), 8 sets (as 7+eaeA), 9 sets (as8+gnd), and such that the final concentration of each probe was 5 nM.

[0290] A total extract of Salmonella enteritidis genomic DNA was addedas a template such that the estimated number of genome equivalents waseither 10⁵, 10⁴, 10³ or zero as a negative control. XL-SDA reactionswere performed as described below, and the reaction products analyzed byboth acrylamide gel electrophoresis and electronic hybridization on amicroelectrode array.

[0291] XL-SDA reactions were performed as follows although theconcentrations and volumes of reaction components and time/temperatureprofiles may be adjusted as necessary. The volumes used assume a 25 μlligation reaction volume and a 50 μl final reaction volume for SDA.

[0292] In a 250 μl microcentrifuge tube, solutions of the followingreagents were combined to give the final concentrations shown: (1) two(or more) target-specific ligation probes (e.g. probe exo-LP1 having a5′ sequence substantially complimentary to a portion of the targetsequence of interest and a 3′ sequence complimentary to a universalamplification primer and probe exo-LP2 having a 3′ end sequencesubstantially complementary to a portion of the target sequence locateddownstream of LP1 and a 5′ end sequence identical to a second universalamplification primer) to give probe concentrations of 5 nM of eachprobe; and, (2) a solution containing the template DNA of interest.

[0293] The exonuclease/ligation reaction was initiated by the additionof the following: a thermostable DNA ligase (such as Taq DNA ligase orPfu DNA ligase); a thermostable DNA polymerase having 5′-3′ exonucleaseactivity, (such as Taq DNA polymerase); buffer salts to give finalconcentrations shown in Table VII below; and dATP at 2.8 mM in a 25 μlreaction. TABLE VII Final concentration in reaction for each ligaseBuffer Taq DNA ligase Pfu DNA ligase Component (1 U/rxn) (0.2 U/rxn)Tris-HCl pH 7.6 10 mM 10 mM Potassium Acetate 25 mM 25 mM MagnesiumAcetate 10 mM 10 mM Dithiothreitol 1 mM 1 mM Nicotinamide adenine 1 mMNONE dinucleotide Adenosine triphosphate NONE 10 μM

[0294] The ligation/exonuclease reaction was incubated at 60° C. for15-30 minutes. Then, 20 μl of an SDA stock mix containing additionalbuffer components, a mixture of dNTPs such that the final reactioncontains all four dNTPs, and amplification primers, is added to give thefinal reaction concentrations (in 50 μl) shown in Table VIII. TABLE VIIIFinal Concentrations in 50 μl SDA Component reaction Potassium phosphate35 mM Bovine serum albumin 80 μg/ml Magnesium acetate 10 mMDeoxynucleotide triphosphates (dGTP, 1.4 mM dC_(αS)TP, TTP)Amplification primers (S1 and S2) 250 nM

[0295] Then, 5 μl's of a solution containing the SDA enzymes is added togive the following final concentrations: BsoB1 restriction enzyme at 0.8enzyme units/μl (40U/rxn) and Bst DNA polymerase at 0.32 enzyme units/μl(16 U/rxn). This reaction mixture is then incubated at 60° C. for 30minutes to allow the SDA reaction to proceed. The reaction is stopped byplacing it on ice and the amplified products are detected.

[0296] The reaction products generated were analyzed by both acrylamidegel electrophoresis and electronic hybridization on a microelectrodearray. An analysis of 5 μl of the XL-SDA reactions by acrylamide gelelectrophoresis demonstrated that specific amplification product is madein a template concentration-dependent manner in all combinations ofligation probes. To demonstrate specific amplification of the Salmonellaenteritidis spaQ gene sequence, the ligation-SDA reaction products wereanalyzed on a microelectrode array where specific capture probes forfive of the bacterial genes are pre-arranged at discrete locations. FIG.24 shows that in all samples analyzed, the spaQ sequence was detected.

[0297] The foregoing is intended to be illustrative of the embodimentsof the present invention, and are not intended to limit the invention inany way. Numerous variations and modifications of the present inventionmay be effected without departing from the true spirit and scope of theinvention. As is understandable to one of ordinary skill in the art,each of the embodiments as disclosed above may be used together in anycombination. For example, SDA may be carried out in connection with anelectronically addressable microchip wherein amplification primersspecific for a target nucleic acid (such as branched or unbranchedprimer pairs having complementary sequence to ligation probes or othertarget nucleic acids of interest) are anchored to an electronicallyaddressable capture pad, target nucleic acid is electronically addressedto such capture pads, and SDA is performed under high pressure. Inanother example, SDA may be carried out in connection with anelectronically addressable microchip wherein allele-specificamplification primers (such as branched or unbranched primer pairs) areanchored to an electronically addressable capture pad, target nucleicacid is electronically addressed to such capture pads, and SDA isperformed under high pressure or in the alternative at atmosphericpressure. In still another combination example, SDA may be carried outin connection with an electronically addressable microchip wherein theSDA reaction is carried out using noncleaveable primers or underasymmetric amplification conditions. Additionally, other combinationsmay include ligation-based SDA in combination with the electronicallyaddressable microchip either under elevated or normal atmosphericpressures. As is understandable to one of ordinary skill in the art,many other combinations are possible.

[0298] Although the invention has been described with respect tospecific modifications, the details thereof are not to be construed aslimitations, for it will be apparent that various equivalents, changesand modifications may be resorted to without departing from the spiritand scope thereof and it is understood that such equivalent embodimentsare to be included herein.

[0299] All publications and patent applications are herein incorporatedby reference to the same extent as if each individual publication orpatent application was specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. A method for the amplification, multiplexassaying, and detection of target nucleic acids of interest using abioelectronic microchip comprising the steps of: a. introducing at leastone of said target nucleic acids of interest onto a bioelectronicmicrochip having a plurality of electronically addressable capturesites; b. amplifying said target nucleic acids to form amplicons of saidtarget nucleic acids wherein said target nucleic acids are amplifiedusing an amplification reaction wherein a multiplicity of sets ofamplification primers are used that include unequal ratios of upstreamand downstream primers; c. capturing said amplicons of said targetnucleic acids onto specified capture sites of said microchip by acapture probe that has specificity for said amplicons; and d. detectingthe presence of said captured amplicons.
 2. The method of claim 1wherein amplification of the target nucleic acid is by stranddisplacement amplification.
 3. The method of claim 1 whereinamplification of the target nucleic acid is by at least one of themethods selected from the group consisting of strand displacementamplification, allele-specific strand displacement amplification,nucleic acid sequence-based amplification, and ligation-based stranddisplacement amplification.
 4. The method of claim 1 wherein saidcapturing of amplicons in (c) is carried out by passive hybridization.5. The method of claim 1 wherein said capturing of amplicons in (c) iscarried out by electronic biasing of said capture sites.
 6. The methodof claim 1 wherein said amplification is carried out in part usingnoncleavable and/or non-extending primers.
 7. The method of claim 1wherein said amplification, multiplex assaying and detecting are carriedout either consecutively or simultaneously in relation to one another.8. The method of claim 1 wherein detection of amplicons is by at leastone of fluorescence, chemiluminescence, and electrochemiluminescence. 9.The method of claim 1 wherein target nucleic acids of interest arederived from a source selected from the group consisting of blood,sputum, stool, tissue, media, semen, hair, urine, food, water, andculture inoculated with at least one of blood, sputum, stool, tissue,media, semen, hair, urine, food, and water.
 10. The method of claim 1wherein the nucleic acid of interest is derived from a source selectedfrom the group consisting of a bacterium, a virus, a human, an animal,or a plant.
 11. The method according to claim 1 wherein at least oneprimer of any one of said sets is in molar excess as compared to anyother primer in such set.
 12. The method according to claim 11 wherein aprimer that is in molar excess in said amplification reaction islabeled.
 13. A method for the amplification, multiplex assaying, anddetection of target nucleic acids of interest using a bioelectronicmicrochip comprising the steps of: a. introducing at least one of saidtarget nucleic acids of interest onto a bioelectronic microchip having aplurality of electronically addressable capture sites; b. addressingsaid target nucleic acids of interest to at least one of said capturesites; c. amplifying said target nucleic acids to form amplicons of saidtarget nucleic acids wherein said target nucleic acids are amplifiedusing an amplification reaction wherein a multiplicity of sets ofamplification primers are used that include unequal ratios of upstreamand downstream primers; d. capturing said amplicons of said targetnucleic acids onto specified capture sites of said microchip by acapture probe that has specificity for said amplicons; and e. detectingthe presence of said captured amplicons.
 14. The method of claim 13wherein amplification of the target nucleic acid is by stranddisplacement amplification.
 15. The method of claim 13 whereinamplification of the target nucleic acid is by at least one of themethods selected from the group consisting of strand displacementamplification, allele-specific strand displacement amplification,nucleic acid sequence-based amplification, and ligation-based stranddisplacement amplification.
 16. The method of claim 13 wherein saidaddressing of said target nucleic acids in (b) is carried out by passivehybridization.
 17. The method of claim 13 wherein said addressing ofsaid target nucleic acids in (b) is carried out by electronic biasing ofsaid capture sites.
 18. The method of claim 13 wherein said capturing ofamplicons in (d) is carried out by passive hybridization.
 19. The methodof claim 13 wherein said capturing of amplicons in (d) is carried out byelectronic biasing of said capture sites.
 20. The method of claim 13wherein said amplification is carried out in part using noncleavableprimers and/or non-extending primers.
 21. The method of claim 13 whereinsaid amplification, multiplex assaying and detecting are carried outeither consecutively or simultaneously in relation to one another. 22.The method of claim 13 wherein detection of amplicons is by at least oneof fluorescence, chemiluminescence, and electrochemiluminescence. 23.The method of claim 13 wherein target nucleic acids of interest arederived from a source selected from the group consisting of blood,sputum, stool, tissue, media, semen, hair, urine, food, water, andculture inoculated with at least one of blood, sputum, stool, tissue,media, semen, hair, urine, food, and water.
 24. The method of claim 13wherein the nucleic acid of interest is derived from a source selectedfrom the group consisting of a bacterium, a virus, a human, an animal,or a plant.
 25. The method according to claim 13 wherein at least oneprimer of any one of said sets is in molar excess as compared to anyother primer in such set.
 26. The method according to claim 25 wherein aprimer that is in molar excess in said amplification reaction islabeled.
 27. A kit for carrying out asymmetric SDA reactions for use ona bioelectronic microchip comprising: a. one or more oligonucleotidesspecific for Factor V, Hemochromotosis, or a bacterium, whicholigonucleotides comprise amplification primers, bumper primers, captureprobes, and/or signal probes selected from the group consisting of Seq.Id. Nos. 1-62.
 28. A kit according to claim 27 wherein said signalprobes are labeled with a detectable label.